Compositions and methods for the treatment or the prevention of e. coli infections and for the eradication or reduction of e. coli surfaces

ABSTRACT

Compositions and methods for treating or preventing  E. coli  infections are provided. The compositions can be formulated as pharmaceutical compositions or as disinfectants, sanitizers, detergents or antiseptics, and can be used to eradicate or reduce  E. coli  populations and thereby treat or prevent infection by  E. coli . The compositions include one or more digestive enzymes, e.g., one or more protease, lipases, and amylases. Methods of use of the compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Applications Ser. Nos. 61/142,718, filed Jan. 6, 2009;61/153,279, filed Feb. 17, 2009; and 61/170,856, filed Apr. 20, 2009,the contents of all of which are hereby incorporated by reference intheir entirety.

TECHNICAL FIELD

This disclosure relates to compositions, including pharmaceuticalcompositions such as antibiotic compositions, and methods of using thesame for treating or preventing E. coli infections in humans and otheranimals. This disclosure also relates to compositions, such asdisinfectants, sanitizers, antiseptics, and detergents, and methods ofusing the same for eradicating or reducing the presence of E. coli. onsurfaces, including inanimate and biological surfaces (e.g., skin,wounds), and/or for attenuating the infectivity of E. coli in order toprevent and/or to reduce the spread of E. coli infections.

BACKGROUND

Escherichia coli (E. coli) is a type of bacteria that can cause severehuman illness. While there are many types of E. coli bacteria, onlycertain types cause food borne illness. Hundreds of harmless strains ofE. coli can be found widely in nature, including the intestinal tractsof humans and other warm-blooded animals. Disease-causing strains,however, are a frequent cause of both intestinal and urinary-genitaltract infections.

In 1982, scientists identified the first harmful food borne strain of E.coli in the United States. The disease-causing food borne E. coli mostcommonly found in this country is called O157:H7. Cattle are the mainsources of E. coli O157:H7, but these bacteria also can be found inother domestic and wild mammals. E. coli serotype O157:H7 is a rarevariety of E. coli that produces large quantities of one or morerelated, potent toxins that cause severe damage to the lining of theintestine.

Several different strains of harmful E. coli can cause diarrhealdisease. Particularly dangerous types of E. coli, such as E. coliO157:H7, produce one or more kinds of toxins (poisons) called Shigatoxins. Shiga toxins can severely damage the lining of intestines andkidneys. These types of bacteria are called Shiga toxin-producing E.coli (STEC). STEC often causes bloody diarrhea and can lead to kidneyfailure in children or in people with weakened immune systems. STEC isalso referred to as verocytotoxic E. coli (VTEC) or enterohemorrhagic E.coli (EHEC); these all refer generally to the same group of bacteria.The most commonly identified STEC in North America is E. coli O157:H7(often shortened to E. coli O157 or even just “O157”).

Enterotoxigenic E. coli (ETEC), which produce a different toxin, cancause diarrhea. These strains typically cause so-called travelers'diarrhea because they commonly contaminate food and water in developingcountries. Enteropathogenic E. coli (EPEC) cause persistent diarrhea(lasting 2 weeks or more) and are more common in developing countrieswhere they can be transmitted to humans through contaminated water orcontact with infected animals. Other types of E. coli, including O26:H11and O111:H8, also have been found in the United States and can causedisease in people.

E. coli infection is characterized by severe cramping (abdominal pain)and diarrhea that is initially watery but becomes grossly bloody.Occasionally vomiting occurs. Fever is either low-grade or absent. Theillness is usually self-limited and lasts for an average of 8 days. Someindividuals exhibit watery diarrhea only.

Hemorrhagic colitis is diagnosed by isolation of E. coli of serotypeO157:H7 or other verotoxin-producing E. coli from diarrheal stools.Alternatively, the stools can be tested directly for the presence ofverotoxin. Confirmation can be obtained by isolation of E. coli of thesame serotype from the incriminated food.

In addition to E. coli O157, many other kinds (called serogroups) ofSTEC cause disease. These other kinds are sometimes called “non-O157STEC.” E. coli serogroups O26, 0111, and O103 are the non-O157serogroups that most often cause illness in people in the United States.

People of any age can become infected with STEC. Very young children andthe elderly are more likely to develop severe illness and hemolyticuremic syndrome (HUS) than others, but even healthy older children andyoung adults can become seriously ill. The symptoms of STEC infectionsvary for each person but often include severe stomach cramps, diarrhea(often bloody), and vomiting.

Around 5 to 10% of those who are diagnosed with STEC infection develop apotentially life-threatening complication known as hemolytic uremicsyndrome (HUS). Clues that a person is developing HUS include decreasedfrequency of urination, feeling very tired, and losing pink color incheeks and inside the lower eyelids. Persons with HUS should behospitalized because their kidneys may stop working and they may developother serious problems. Most persons with HUS recover within a fewweeks, but some suffer permanent damage or die.

STEC live in the guts of ruminant animals, including cattle, goats,sheep, deer, and elk. The major source for human illnesses is cattle.STEC that cause human illness generally do not make animals sick. Otherkinds of animals, including pigs and birds, sometimes acquire STEC fromthe environment and may carry or spread it.

SUMMARY

This disclosure relates to the prevention and/or treatment of E. coliinfections, including antibiotic-resistant and virulent E. coliinfections, such as STEC, ETEC, or EPEC, with the use of apharmaceutical composition comprising one or more digestive enzymes,such as pancreatic or other digestive-track enzymes (e.g., porcinepancreatic enzymes) or plant-, fungal-, or microorganism-derivedenzymes, that break down components of food. As used herein, apharmaceutical composition can be used for human or veterinaryindications. Accordingly, the pharmaceutical compositions can be usefulfor prophylactic and/or therapeutic treatment of human or othermammalian populations (e.g., pig, horse, cow, sheep, goat, monkey, rat,mouse, cat, dog) or of bird populations (e.g., duck, goose, chicken,turkey).

The pharmaceutical compositions can be used on their own, and/or incombination with other antibacterial or antibiotic (e.g., anti-E. coli)regimens, and/or with other therapeutic or antibiotic agentspost-infection to treat E. coli infections.

Also provided herein are bacteriocidal and/or bacteriostaticcompositions comprising one or more digestive enzymes for use as or indisinfectants, sanitizers, detergents, and antiseptics, e.g., inhospitals, nursing homes, nurseries, daycares, schools, workenvironments, food service settings, public transportation and restroomfacilities, to reduce, attenuate, and/or destroy E. coli present in suchsettings. The surfaces treated with the described compositions can belarge (e.g., operating room tables, doors, changing tables, ventilationsystems) or small (e.g., medical devices, door handles); inanimate ornon-living (tables) or living tissue (hands, e.g., detergents forhand-washing; wounds, e.g., surgical wounds or wounds resulting fromaccidents/trauma). The compositions can thus be useful to treat surfacesto reduce or eradicate E. coli thereon, or to attenuate or reduce theinfectivity of E. coli, and thereby prevent or reduce the spread of E.coli.

Accordingly, it is an object of the present disclosure to provide amethod for the treatment or prevention of E. coli infection in a bird ora mammal, comprising administering to the bird or mammal atherapeutically effective amount of a pharmaceutical compositioncomprising one or more digestive enzymes. In some embodiments, the oneor more digestive enzymes comprise one or more enzymes selected from thegroup consisting of proteases, amylases, celluloses, sucrases, maltases,papain, and lipases. In some embodiments, the one or more digestiveenzymes comprise one or more pancreatic enzymes. The one or moredigestive enzymes are, independently, derived from an animal source, amicrobial source, a plant source, a fungal source, or are syntheticallyprepared. In some embodiments, the animal source is a pig pancreas.

In some embodiments, a pharmaceutical composition comprises at least oneamylase, a mixture of proteases comprising chymotrypsin and trypsin, andat least one lipase. In some embodiments, a pharmaceutical compositioncomprises at least one protease and at least one lipase, and wherein theratio of total proteases to total lipases (in USP units) ranges fromabout 1:1 to about 20:1.

In some embodiments, the pharmaceutical composition is a dosageformulation selected from the group consisting of: pills, tablets,capsules, caplets, sprinkles, creams, lotions, aerosols, emulsions,powders, liquids, gels, and a combination of any thereof.

In some embodiments, the pharmaceutical composition is formulated fororal administration, or for topical administration, or for intranasal,or for transmucosal administration.

Also provided is a method of treating a mammal or bird exhibiting one ormore symptoms of an E. coli infection comprising administering to themammal or bird a therapeutically effective amount of a compositioncomprising one or more digestive enzymes.

Further provided is a method for treating diarrhea comprisingadministering a pharmaceutical composition comprising one or moredigestive enzymes to an individual. The disclosure also features amethod for sanitizing or disinfecting a surface to reduce the amount ofE. coli thereon or to eradicate the E. coli thereon, comprising applyingto the surface a composition comprising one or more digestive enzymes.The surface can be a living surface (e.g., skin, wound) or an inanimateor non-living surface (e.g., medical device, food product).

Also provided herein is a method for reducing the amount of E. colipresent on a skin region, tissue, or wound of a mammal or birdcomprising applying to the skin region, tissue, or wound a compositioncomprising one or more digestive enzymes.

Also featured is a disinfectant comprising one or more digestiveenzymes, wherein the disinfectant has a phenol coefficient of >1 toabout 20 for S. aureus or E. coli.

The disclosure also provides an antibiotic comprising one or moredigestive enzymes, wherein the antibiotic is bacteriocidal and/orbacteriostatic for E. coli.

Similarly, a detergent comprising one or more digestive enzymes is alsoprovided, wherein the detergent is bacteriocidal and/or bacteriostaticfor E. coli.

Also provided is an antiseptic comprising one or more digestive enzymes,wherein the antiseptic is bacteriocidal and/or bacteriostatic for E.coli.

The disclosure also provides a disinfectant comprising one or moredigestive enzymes, wherein the disinfectant is bacteriocidal and/orbacteriostatic for E. coli.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Any referencedisclosed herein, such as a patent, patent application, specification,book, article, or scientific publication, is incorporated by referencein its entirety. Other features, objects, and advantages of theinvention will be apparent from the description and drawings, and fromthe claims.

DETAILED DESCRIPTION

The term “administration” or “administering” refers to a method ofgiving a dosage of a composition or pharmaceutical composition to avertebrate or invertebrate, including a mammal, a bird, a fish, or anamphibian, where the method is by any route, e.g., intrarespiratory,nasal, topical, oral, intravenous, intraperitoneal, intramuscular,transmucosal, buccal, rectal, vaginal, or sublingual. The preferredmethod of administration can vary depending on various factors, e.g.,the components of the pharmaceutical composition, the site of thedisease, the disease involved, and the severity of the disease.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, dogs, and cats, but alsoincludes many other species.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, isotonic and absorption delaying agents and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredients, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients, such asantibiotics, antifungals, antimicrobials, can also be incorporated intothe compositions. In addition, various adjuvants such as are commonlyused in the art may be included. These and other such compounds aredescribed in the literature, e.g., in the Merck Index, Merck & Company,Rahway, N.J. Considerations for the inclusion of various components inpharmaceutical compositions are described, e.g., in Gilman et al. (Eds.)(2006); Goodman and Gilman's: The Pharmacological Basis of Therapeutics,11th Ed., The McGraw-Hill Companies.

“Subject” or “patient” or “individual” as used herein, means a human ora non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep,a pig, a goat, a non-human primate or a bird, e.g., a chicken, as wellas any other vertebrate or invertebrate.

By “therapeutically effective amount” or “pharmaceutically effectiveamount” is typically one which is sufficient to achieve the desiredeffect and may vary according to the nature and severity of the diseasecondition, the nature of the subject, and the potency of thecomposition. It will be appreciated that different concentrations may beemployed for prophylaxis than for treatment of an active disease. Thisamount can further depend upon the patient's height, weight, sex, ageand medical history.

A therapeutic effect relieves, to some extent, one or more of thesymptoms of the disease, and includes curing a disease. “Curing” meansthat the symptoms of active disease are eliminated. However, certainlong-term or permanent effects of the disease may exist even after acure is obtained (such as tissue damage).

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a pharmaceutical composition for therapeutic purposes. Theterm “therapeutic treatment” refers to administering treatment to apatient already suffering from a disease thus causing a therapeuticallybeneficial effect, such as ameliorating existing symptoms, preventingadditional symptoms, ameliorating or preventing the underlying metaboliccauses of symptoms, postponing or preventing the further development ofa disorder and/or reducing the severity of symptoms that will or areexpected to develop.

The present disclosure provides compositions comprising one or moredigestive enzymes and methods of using the same for the treatment and/orprevention of E. coli infections, including antibiotic-resistant formsof E. coli and virulent forms such as STEC, ETEC, or EPEC. The presentdisclosure also provides compositions comprising one or more digestiveenzymes and methods of using the same as antiseptics, detergents,disinfectants, and sanitizers, e.g., as bacteriocidal and/orbacteriostatic compositions, to eradicate or attenuate the E. coliand/or to reduce its infectivity. The compositions described hereininclude one or more digestive enzymes, which are postulated to assist inthe reduction, weakening, or eradication of E. coli, and thus to preventcontraction of E. coli infections or to treat E. coli infections (e.g.,improve or ameliorate the symptoms or reduce the time course of theinfection).

Compositions

A composition for use as described herein can include one or moredigestive enzymes. While not being bound by theory, it is believed thatthe digestive enzyme(s) in the composition can degrade E. coli cellwall, membrane, and/or protein structures, leading to the bacteriostaticand/or bacteriocidal activity. The compositions demonstratespecies-specific bacteriocidal/bacteriostatic activity against S. aureusand E. coli, but not against S. enterica, possibly demonstrating thatthe vulnerability of the two organisms derives from proteolyticdegradation of a similar protein sequence present in the two organisms.

A digestive enzyme as described herein is an enzyme that can break downone or more components of food (e.g., proteins, fats, carbohydrates).The digestive enzymes can be animal-derived (e.g., pancreatic or otherdigestive-track enzymes), or plant-, fungal-, or microorganism-derivedenzymes, or can be synthetically prepared. Many digestive enzymes arecommercially available or can be isolated and purified from othersources by methods well known to those having ordinary skill in the art.Enzymatic activity of the enzymes can also be evaluated using standardassays.

The digestive enzymes can be used in any combination of type of enzymeand any combination of enzyme sources. In some embodiments, the one ormore digestive enzymes comprise one or more enzymes selected from thegroup consisting of proteases, amylases, celluloses, sucrases, maltases,papain (e.g., from papaya), bromelain (e.g., from pineapple),hydrolases, and lipases. In some embodiments, the one or more digestiveenzymes comprise one or more pancreatic enzymes. In some embodiments,the composition comprises one or more proteases, one or more lipases,and one or more amylases. In some embodiments, the one or more proteasescomprise chymotrypsin and trypsin. In some embodiments, a composition asdescribed herein consists essentially of, or consists of, the one ormore digestive enzymes.

In certain embodiments, the composition can comprise at least oneamylase, at least two proteases, and at least one lipase. In certainembodiments, the composition can further include one or more hydrolases,papain, bromelain, papaya, celluloses, pancreatin, sucrases, andmaltases.

As indicated, the one or more digestive enzymes can be derived from ananimal source. In some embodiments, the animal source is a pig, e.g., apig pancreas. Pig pancreatic enzyme extracts and formulations are knownto those having ordinary skill in the art and are commercially availableor can be prepared using known methods. For example, a pancreatic enzymecomposition can be purchased from Scientific Protein Laboratories(designated PEC). A pancreatic enzyme composition, or any compositionherein, can be adjusted to modify the amount of one or more digestiveenzymes contained therein, e.g., the lipase, amylase, or proteasecontent, such as by production and/or processing methods or by theselective addition of exogenous enzymes, activators, or inhibitors tothe composition.

In certain circumstances, it may be desirable to have relatively higheractivity of proteases than lipases. Thus, in some embodiments, acomposition comprises at least one protease and at least one lipase,wherein the ratio of total proteases to total lipases (in USP units)ranges from about 1:1 to about 20:1 including 1:1, 2:1, 3;1, 4:1, 5;1,6:1, 7:1, 8:1, 9:1, 10:1, 11;1, 12;1, 13;1, 14:1, 15:1, 16;1, 17:1,18:1, 19:1 and 20:1, long with all values in-between. In someembodiments, the ratio of proteases to lipases ranges from about 4:1 toabout 10:1 including 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1, along withall values in-between.

In certain circumstances it may be useful to modify the amount of aparticular enzymatic activity in a given composition. The activity ofthe one or more digestive enzymes can be adjusted in a variety of waysknown to the skilled artisan, e.g., by increasing the amount of theparticular enzyme, or by adjusting the components of the composition,e.g., via the use of stabilizers, inhibitors, and activators. In someembodiments, a composition described herein includes one or moreproteases having an activity of from about 0.05 to about 400 USP Unitsper mg of the composition, or any value therebetween (e.g., 0.1; 0.2;0.25; 0.5; 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75,100, 150, 200, 250, 300, 350 USP Units per mg). In some embodiments, acomposition described herein includes one or more lipases having anactivity of from about 0.005 to about 50 Units per mg of thecomposition, or any value therebetween (e.g., 0.01, 0.02, 0.025, 0.03,0.04, 0.05, 0.06, 0.08, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 14, 16, 18, 20, 22, 25, 28, 30, 35, 38, 40, 45 USP Units per mg). Insome embodiments, a composition described herein includes one or moreamylases having an activity of from about 0.05 to about 400 USP Unitsper mg of the composition, or any value therebetween (e.g., 0.1; 0.2;0.25; 0.5; 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75,100, 150, 200, 250, 300, 350 USP Units per mg). In some embodiments, acomposition described herein includes one or more proteases in the aboveactivity range, one or more lipases in the above activity range, and oneor more amylases in the above activity range. One exemplary embodimentincludes one or more proteases having an activity in the range of about150-250 USP units/mg; one or more lipases having an activity in therange of about 20-40 USP units/mg; and one or more amylases having anactivity in the range of about 200-300 USP units/mg.

In some embodiments, a composition can be formulated so as to stabilizethe one or more digestive enzymes, e.g., to preserve the enzymaticactivity of the enzymes. Stabilization techniques can limit or preventauto-degradation of the one or more enzymes in a composition and helpmaintain enzymatic activity, increase shelf-life, and aid in thetolerance of the activity of the compositions to changes in temperature,humidity, and storage conditions. For example, in some embodiments, theone or more enzymes in the composition are encapsulated, e.g.,lipid-encapsulated. In other applications, variations in excipients, pH,enzyme inhibitors, etc. can be employed to aid in stabilizing theenzymes. Appropriate stabilization techniques will depend on theintended application for the composition (e.g., antibiotic vs.detergent), the route of administration, the form of the composition,the intended site of delivery/activity, and other factors, and can bedetermined by those having ordinary skill in the art.

Certain useful enzyme activity stabilizers include compounds thatprovide a source of free calcium in a solution such as for examplecalcium salts; alkyl or branched alcohols such as for example ethanoland isopropyl alcohol; alkanolamines such as for exampletriethanolamine; acids, such as organic acids; and mixtures of petroleumdistillates.

In certain embodiments, an enzyme activity stabilizer can be acomposition selected from (1) compositions known to be effective instabilizing enzymes in liquid aqueous solutions, including enzymestabilizing compounds and systems, (2) selected “micelle inhibitors”,and mixtures of (1) and (2). In some embodiments, the activitystabilizer is a suitable concentration of boron anions. In some cases,the activity stabilizer is solvated in a polyol and may be combined withenzyme stabilizing synergists or adjuvants forming an enzyme stabilizingsystem. Preferred “micelle inhibitors” include species known to modifyas well as to inhibit micelle formation and may be selected from watermiscible solvents such as C1-C6 alkanols, C1-C6 diols, C2-C24 alkyleneglycol ethers, alkylene glycol alkyl ethers, and mixtures thereof. Ahighly preferred micelle inhibitor is di-(propylene glycol) methyl ether(“DPM”) and analogues thereof which modify micelle formation.

One example of an “enzyme stabilizing system” is a boron compound (e.g.boric acid) which in the past has been used alone or with selected otheradjuvants and or synergists (e.g. polyfunctional amino compounds,antioxidants, etc) to protect proteolytic and other enzymes in storageand in various products.

An activity stabilizer may be chosen to substantially minimize theMinimum Inhibitory Concentration (“MIC”) of digestive enzyme in theformulation. MIC is a measure of the minimum concentration of thebiocide which succeeds in preventing bacterial growth in a cultureduring a specified time period, for example 24 hrs. Details of the MICtest are shown in “Bailey & Scott ‘Diagnostic Microbiology’, 8thedition, 1990 at page 177.

In some embodiments, a composition described herein can be coated with avariety or natural or synthetic coatings, e.g., to provide timed releaseof the enzymes, to provide flavor or odor masking, or to stabilize theenzymes. Coated enzyme preparations, including lipid-coated orlipid-encapsulated enzyme compositions, comprising one or more digestiveenzymes useful for the methods and compositions described herein aredisclosed in U.S. Ser. No. 12/386,051, filed Apr. 13, 2009, incorporatedherein by reference in its entirety. Such coated preparations canprovide desired features, including increased shelf stability, reducedaerosolization of powder or solid formulations, odor and taste-masking,enzyme stabilization, and delayed or timed release of the enzymes.

Other additives for inclusion in the compositions described herein canbe determined by those having ordinary skill in the art, and will bebased on a number of features, including intended application, e.g.,human vs. veterinary applications; desired release profile; desiredpharmacokinetics; safety; stability; and physical characteristics(smell, color, taste, pour, aerosilization). Suitable formulationingredients, excipients, binders, bulking agents, flavorants, colorants,etc. can be determined and evaluated by methods known to those havingordinary skill.

Pharmaceutical Compositions and Antibiotics for Human or Veterinary Use

Compositions described herein can be formulated as pharmaceuticalcompositions, e.g., can include a composition as described previouslyformulated with one or more pharmaceutically acceptable carriers orexcipients. The pharmaceutical compositions are useful for treating orpreventing E. coli infections in humans and other animals, such asmammals (e.g., cows, horses, pigs, sheep, goat, monkey, cats, dogs,mice, rats) and birds (chickens, turkeys, ducks, geese). Apharmaceutical composition for treating E. coli infections can also bereferred to as an antibiotic or antibiotic composition herein.

The susceptibility of E. coli, including virulent and resistant forms,to an antibiotic composition described herein can be determined bymethods known to those having ordinary skill in the art. One rapidprocedure uses commercially filter paper disks that have beenimpregnated with a specific quantity of the antibiotic composition.These disks are placed on the surface of agar plates that have beenstreaked with a culture of the E. coli being tested, and the plates areobserved for zones of growth inhibition. The broth dilutionsusceptibility test involves preparing test tubes containing serialdilutions of the composition in liquid culture media, then inoculatingthe organism being tested into the tubes. The lowest concentration ofdrug that inhibits growth of the bacteria after a suitable period ofincubation is reported as the minimum inhibitory concentration (MIC).

The resistance or susceptibility of E. coli to an antibiotic describedherein can be determined on the basis of clinical outcome, i.e., whetheradministration of that antibiotic to a subject infected by that organismwill successfully cure the subject. Alternatively, to facilitate theidentification of antibiotic resistance or susceptibility using in vitrotest results, the National Committee for Clinical Laboratory Standards(NCCLS) has formulated standards for antibiotic susceptibility thatcorrelate clinical outcome to in vitro determinations of the minimuminhibitory concentration of antibiotic.

Administration of the pharmaceutical compositions herein can be via anyof the accepted modes of administration for agents that serve similarutilities including, but not limited to, orally, subcutaneously,intravenously, intranasally, topically, transdermally, transmucosally,intraperitoneally, intramuscularly, intrapulmonarilly, vaginally,rectally, or intraocularly. Oral, transmucosal, topical, and parenteraladministrations, for example, are customary in treating E. coliinfection indications.

In the pharmaceutical compositions, effective concentrations of one ormore digestive enzymes are mixed with a suitable pharmaceuticalexcipient or carrier. The concentrations of the digestive enzymes in thecompositions are effective for delivery of an amount, uponadministration, that is useful in the reduction or eradication of E.coli bacteria, and/or to treat or ameliorate of one or more of thesymptoms associated with E. coli infection.

Antiobiotic compositions can be formulated for single dosageadministration. To formulate a composition, the weight fraction ofdigestive enzymes is dissolved, suspended, dispersed or otherwise mixedin a selected carrier at an effective concentration such that thebacteria are reduced or eradicated, the treated condition is relieved,or one or more symptoms are ameliorated.

The digestive enzymes are included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the digestive enzymes in in vitro and in vivo, and thenextrapolated therefrom for dosages for humans.

The concentration of digestive enzymes in the pharmaceutical compositionwill depend on absorption, inactivation and excretion rates of theenzymes, the physicochemical characteristics of the enzymes, the dosageschedule, the dosage form, and amount administered as well as otherfactors known to those of skill in the art.

The pharmaceutical composition may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

Upon mixing or addition of the digestive enzymes, the resulting mixturemay be a solution, suspension, gel, powder, emulsion or the like. Theform of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thedigestive enzymes in the selected carrier or vehicle.

Compositions intended for pharmaceutical use may be administered ascrystalline or amorphous products. Pharmaceutically acceptablecompositions include solid, semi-solid, liquid, gel, powder, and aerosoldosage forms, such as, e.g., tablets, capsules, caplets, sprinkles,powders, liquids, suspensions, emulsions, gels, suppositories, aerosolsor the like. They may be obtained, for example, as films by methods suchas precipitation, crystallization, freeze drying, spray drying, orevaporative drying. Microwave or radio frequency drying may be used forthis purpose. The compositions can also be administered in sustained orcontrolled release dosage forms, including depot injections, osmoticpumps, pills, specialized coatings (e.g., enteric coatings) on oraldosage forms, transdermal (including electrotransport) patches, and thelike, for prolonged and/or timed, pulsed administration at apredetermined rate. In some embodiments, the compositions are providedin unit dosage forms suitable for single administration of a precisedose.

The compositions can be administered either alone or more typically incombination with a conventional pharmaceutical carrier, excipient or thelike. The term “excipient” is used herein to describe any ingredientother than the compound(s) (enzymes) used in the composition.Pharmaceutically acceptable excipients include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifyingdrug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol1000 succinate, surfactants used in pharmaceutical dosage forms such asTweens or other similar polymeric delivery matrices, serum proteins,such as human serum albumin, buffer substances such as phosphates,glycine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes, such asprotamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium-chloride, zinc salts, colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, cellulose-based substances,polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, and wool fat.Cyclodextrins such as α-, β, and γ-cyclodextrin, or chemically modifiedderivatives such as hydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-b-cyclodextrins, or other solubilized derivatives canalso be advantageously used to enhance delivery of compositionsdescribed herein. Actual methods of preparing such dosage forms areknown, or will be apparent, to those skilled in this art; for example,see Remington: The Science and Practice of Pharmacy, 21st Edition(Lippincott Williams & Wilkins. 2005).

In one preferred embodiment, the compositions will take the form of aunit dosage form such as a pill or tablet and thus the composition maycontain, along with the active ingredient, a diluent such as lactose,sucrose, dicalcium phosphate, or the like; a lubricant such as magnesiumstearate or the like; and a binder such as starch, gum acacia,polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or thelike. In another solid dosage form, a powder, solution or suspension(e.g., in propylene carbonate, vegetable oils or triglycerides) isencapsulated in a gelatin capsule. Unit dosage forms in which two ormore ingredients are physically separated are also contemplated; e.g.,capsules with granules of enzyme(s) and granules of other ingredients;two-layer tablets; two-compartment gel caps, etc.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. one or more digestive enzymesand optional pharmaceutical adjuvants in a carrier (e.g., water, saline,aqueous dextrose, glycerol, glycols, ethanol or the like) to form asolution or suspension. If desired, the pharmaceutical composition canalso contain minor amounts of nontoxic auxiliary substances such aswetting agents, emulsifying agents, solubilizing agents, pH bufferingagents and the like (e.g., sodium acetate, sodium citrate, cyclodextrinederivatives, sorbitan monolaurate, triethanolamine acetate,triethanolamine oleate, and the like). Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, asemulsions, or in solid forms suitable for dissolution or suspension inliquid prior to injection.

Solid compositions can be provided in various different types of dosageforms, depending on the physicochemical properties of the enzymes, thedesired dissolution rate, cost considerations, and other criteria. Inone of the embodiments, the solid composition is a single unit. Thisimplies that one unit dose of the drug is comprised in a single,physically shaped solid form or article. In other words, the solidcomposition is coherent, which is in contrast to a multiple unit dosageform, in which the units are incoherent.

Examples of single units which may be used as dosage forms for the solidcomposition include tablets, such as compressed tablets, film-likeunits, foil-like units, wafers, lyophilized matrix units, and the like.In one embodiment, the solid composition is a highly porous lyophilizedform. Such lyophilizates, sometimes also called wafers or lyophilizedtablets, are particularly useful for their rapid disintegration, whichalso enables the rapid dissolution of the active compound.

On the other hand, for some applications the solid composition may alsobe formed as a multiple unit dosage form. Examples of multiple units arepowders, sprinkles, granules, microparticles, microcapsules, pellets,beads, lyophilized powders, and the like. In one embodiment, the solidcomposition is a lyophilized powder. Such a dispersed lyophilized systemcomprises a multitude of powder particles, and due to the lyophilizationprocess used in the formation of the powder, each particle has anirregular, porous microstructure through which the powder is capable ofabsorbing water very rapidly, resulting in quick dissolution. In anotherembodiment, the solid composition is a sprinkle formulation.

Another type of multiparticulate system which is also capable ofachieving rapid drug dissolution is that of powders, granules, orpellets from water-soluble excipients which are coated with thecomposition ingredients (e.g., enzymes), so that the enzymes are locatedat the outer surface of the individual particles. In this type ofsystem, the water-soluble low molecular weight excipient is useful forpreparing the cores of such coated particles, which can be subsequentlycoated with a coating composition comprising the enzyme(s) and,preferably, one or more additional excipients, such as a binder, a poreformer, a saccharide, a sugar alcohol, a film-forming polymer, aplasticizer, or other excipients used in pharmaceutical coatingcompositions.

Appropriate dosages for treating or preventing E. coli infections willdepend on the patient (species, age, weight, health), the severity ofthe disease, the strain of the E. coli present, the type of formulation(e.g., liquid or ointment) and other factors known to those havingordinary skill in the art. It is to be noted that concentrations anddosage values may vary with the severity of the condition to bealleviated. It is to be further understood that for any particularpatient, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions.

In some embodiments, the pharmaceutical composition comprises per dose:amylases from about 10,000 to about 60,000 U.S.P, including 10,000,15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000,and 60,000 U.S.P, along with all values in-between, proteases from about10,000 to about 70,000 U.S.P, including 10,000, 15,000, 20,000, 25,000,30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, and70,000, along with all values in-between, and lipases from about 4,000to about 30,000 U.S.P, including, 4,000, 5,000, 10,000, 15,000, 20,000,25,000, and 30,000, along with all values in-between. A pharmaceuticalcomposition can include one or more of : chymotrypsin from about 2 toabout 5 mg including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg, alongwith all values in-between; trypsin from about 60 to about 100 mgincluding 50, 65, 70, 75, 80, 85,90, 95, and 100 mg, including allvalues in between; papain from about 3,000 to about 10,000 USP unitsincluding 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000USP, along with all values in between; and papaya from about 30 to about60 mg, including 30, 35, 40, 45, 50, 55, and 60 mg, along with allvalues in between.

Additional information on particular dosage forms of the compositions isprovided below.

1. Compositions for Oral Administration

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms are tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric-coated, sugar-coated or film-coated. Capsules maybe hard or soft gelatin capsules, while granules and powders may beprovided in non-effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

-   -   a. Solid Compositions for Oral Administration

In certain embodiments, the formulations are solid dosage forms, in oneembodiment, capsules or tablets. The tablets, pills, capsules, trochesand the like can contain one or more of the following ingredients, orcompounds of a similar nature: a binder; a lubricant; a diluent; aglidant; a disintegrating agent; a coloring agent; a sweetening agent; aflavoring agent; a wetting agent; an emetic coating; and a film coating.Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, molasses,polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

The digestive enzymes could be provided in a composition that protectsit from the acidic environment of the stomach. For example, thecomposition can be formulated in an enteric coating that maintains itsintegrity in the stomach and releases the digestive enzymes in theintestine. The composition may also be formulated in combination with anantacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The digestive enzymes can also beadministered as a component of an elixir, suspension, syrup, wafer,sprinkle, chewing gum or the like. A syrup may contain, in addition tothe active digestive enzymes, sucrose as a sweetening agent and certainpreservatives, dyes and colorings and flavors.

The digestive enzymes can also be mixed with other active materialswhich do not impair the desired action, or with materials thatsupplement the desired action, such as antacids, H₂ blockers, anddiuretics. Higher concentrations, up to about 98% by weight of thedigestive enzymes may be included.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the digestive enzymes. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

-   -   b. Liquid Compositions for Oral Administration

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicacid, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Sweetening agents include sucrose, syrups, glycerin andartificial sweetening agents such as saccharin. Wetting agents includepropylene glycol monostearate, sorbitan monooleate, diethylene glycolmonolaurate and polyoxyethylene lauryl ether. Organic acids includecitric and tartaric acid. Sources of carbon dioxide include sodiumbicarbonate and sodium carbonate. Coloring agents include any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include natural flavors extracted from plants suchfruits, and synthetic blends of compounds which produce a pleasant tastesensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is in oneembodiment encapsulated in a gelatin capsule. Such solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, thesolution, e.g., for example, in a polyethylene glycol, may be dilutedwith a sufficient quantity of a pharmaceutically acceptable liquidcarrier, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the digestive enzymes in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. RE28,819 and4,358,603. Briefly, such formulations include, but are not limited to,those containing digestive enzymes provided herein, a dialkylated mono-or poly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

2. Injectables, Solutions and Emulsions

Parenteral administration, in one embodiment characterized by injection,either subcutaneously, intramuscularly or intravenously is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) is also contemplated herein. Briefly, digestive enzymesprovided herein are dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The digestive enzymes diffuse through the outer polymeric membrane in arelease rate controlling step. The percentage of digestive enzymescontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the digestive enzymeor mixture thereof and the needs of the subject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN® 80). A sequestering or chelatingagent of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles; and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing digestive enzymes is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing digestive enzymes that can be injected asnecessary to produce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In oneembodiment, a therapeutically effective dosage is formulated to containa concentration of at least about 0.1% w/w up to about 90% w/w or more,in certain embodiments more than 1% w/w of the digestive enzymes to thetreated tissue(s).

The digestive enzymes may be suspended in micronized or other suitableform or may be derivatized to produce a more soluble active product. Theform of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thedigestive enzymes in the selected carrier or vehicle. The effectiveconcentration is sufficient for ameliorating the symptoms of thecondition and may be empirically determined.

3. Lyophilized Powders

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They may also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving digestiveenzymes as provided herein in a suitable solvent. The solvent maycontain an excipient which improves the stability or otherpharmacological component of the powder or reconstituted solution,prepared from the powder. Excipients that may be used include, but arenot limited to, dextrose, sorbital, fructose, corn syrup, xylitol,glycerin, glucose, sucrose or other suitable agent. The solvent may alsocontain a buffer, such as citrate, sodium or potassium phosphate orother such buffer known to those of skill in the art at, in oneembodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the digestive enzymes. The lyophilized powder can be storedunder appropriate conditions, such as at about 4° C. to roomtemperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selecteddigestive enzymes. Such amount can be empirically determined.

4. Topical Administration

Topical mixtures can be prepared as described for local and systemicadministration. The resulting mixture may be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, powders, solutions, elixirs, lotions, suspensions, tinctures,pastes, foams, aerosols, irrigations, sprays, suppositories, bandages,dermal patches or any other formulations suitable for topicaladministration.

The digestive enzymes may be formulated as aerosols for topicalapplication, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126,4,414,209, and 4,364,923, which describe aerosols for delivery of asteroid useful for treatment of inflammatory diseases, particularlyasthma). These formulations for administration to the respiratory tractcan be in the form of an aerosol or solution for a nebulizer, or as amicrofine powder for insufflation, alone or in combination with an inertcarrier such as lactose. In such a case, the particles of theformulation will, in one embodiment, have diameters of less than 50microns, in one embodiment less than 10 microns.

The digestive enzymes may be formulated for local or topicalapplication, such as for topical application to the skin and mucousmembranes, such as in the eye, in the form of gels, creams, and lotionsand for application to the eye or for intracisternal or intraspinalapplication. Topical administration is contemplated for transdermaldelivery and also for administration to the eyes or mucosa, or forinhalation therapies. Nasal solutions of the digestive enzymes alone orin combination with other pharmaceutically acceptable excipients canalso be administered.

These solutions, particularly those intended for ophthalmic use, may beformulated as 0.01%-10% isotonic solutions, pH about 5-7, withappropriate salts.

Powders can be formed with the aid of any suitable powder base, e.g.,talc, lactose, starch, and the like. Solutions can be formulated with anaqueous or non-aqueous base, and can also include one or more dispersingagents, suspending agents, solubilizing agents, and the like. Topicalgels are prepared using polymers having a molecular weight and level ofconcentration effective to form a viscous solution or colloidal gel ofan aqueous or non-aqueous solution or suspension of digestive enzymes.Polymers from which topical gels may be prepared includepolyphosphoesters, polyethylene glycols, high molecular weightpoly(lactic) acids, hydroxypropyl celluloses, chitosan, polystyrenesulfonates, and the like.

Ointments, creams and lotions are formulated, for example, with anaqueous or oily base and addition of a suitable thickening agent,gelling agent, stabilizing agent, emulsifying agent, dispersing agent,suspending agent, or consistency regulating agent, and the like. Basesinclude water, an alcohol or an oil, such as liquid paraffin, mineraloil, or a vegetable oil, such as peanut or castor oil. Thickening agentsthat can be used according to the nature of the base include softparaffin, aluminum stearate, cetostearyl alcohol, propylene glycol,polyethylene glycols, polyphosphoesters, poly(lactic acids),hydroxyethyl celluloses, hydroxypropyl celluloses, cellulose gums,acrylate polymers, hydrophilic gelling agents, chitosan, polystyrenesulfonate, petrolatum, woolfat, hydrogenated lanolin, beeswax, and thelike.

The ointments, pastes, creams, gels, and lotions can also containexcipients, such as animal and vegetable fats, oils, waxes, paraffins,starch, tragacanth, cellulose derivatives, polyethylene glycols,silicones, bentonites, silicic acid, talc, zinc oxide, and mixturesthereof. Powders and sprays can also contain excipients such as silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Solutions, suspensions or dispersions canbe converted into aerosols or sprays by any of the known means routinelyused for making aerosols for topical application. In general, suchmethods comprise pressurizing or providing a means of pressurizing acontainer of a solution, suspension or dispersion, usually with an inertcarrier gas, and passing the pressured gas through a small orifice.Sprays and aerosols can also contain customary propellants, e.g.,chlorofluorohydrocarbons or volatile unsubstituted hydrocarbons, such asbutane and propane.

Excipients include compounds that promote skin absorption, such asdimethyl sulfoxide (DMSO), partial glycerides of fatty acids, and thelike, present at levels up to about 10 wt % of the total formula weight.Examples of partial fatty acid glycerides include, but are not limitedto IMWITOR 742 and IMWITOR 308 available from SASOL North America, Inc.of Houston, Tex. The topical formulations may also optionally includeinactive ingredients to improve cosmetic acceptability, including butnot limited to, humectants, surfactants, fragrances, coloring agents,emollients, fillers, and the like.

The topical compositions may also include other antibiotic agents,examples of which include bacitracin, neomycin, polymixin, beta-lactams,including penicillin, methicillin, moxalactam and cephalosporins, suchas cefaclor, cefadroxil, cefamandole nafate, cefazolin, cefixime,cefinetazole, cefonioid, cefoperazone, ceforanide, cefotanme,cefotaxime, cefotetan, cefoxitin, cefpodoxime proxetil, ceftazidime,ceftizoxime, ceftriaxone, cefriaxone, cefuroxime, cephalexin,cephalosporin C, cepahlosporin C sodium salt, cephalothin, cephalothinsodium salt, cephalothin dihydrate, cephapirin, cephradine,cefuroximeaxetil, loracarbef, and the like. Essentially anyanti-infective/antibiotic agent that is effective when applied topicallycan be used. Thus, the methods of the present invention for bothtreating active infections and decolonizing skin pathogen populationsinclude methods in which digestive enzymes are applied singularly or incombination, either with no other anti-infective agent, or with at leastone other anti-infective agent.

The topical compositions can be administered directly by the dusting ofa powder, spraying of an aerosol or by spreading a film of an ointment,cream, lotion, solution or gel to the desired area of the skin using thefingertips of the patient or a healthcare provider or other conventionalapplication such as a swab or wipe. The product may be first applied tothe skin and spread with the fingertips or an applicator or applied tothe fingertips and spread over the skin. The compositions may alsooptionally first be coated on the surface of a topical applicator, suchas a bandage, swab, moist woven or non-woven wipe and the like, which isthen applied to the portion of the skin to receive the composition.

The topical compositions of the present invention can be prepared withbase formulations that are essentially conventional to one of ordinaryskill in the art with respect to the ingredients employed, quantitiesthereof, and methods of preparation, all of which require no furtherdescription. Topical compositions according to the present invention canalso be prepared as a cream or lotion based on an emulsion formulationpossessing heretofore unrecognized bactericidal activity, in addition togood skin compatibility and wound-healing properties that isparticularly well-suited for formulation with digestive enzymes.

As discussed above, the present invention is not limited to topicalcream or lotion formulations. Topical formulations based on conventionalsprays, mists, aerosols, lotions, creams, aqueous and non-aqueoussolutions or liquids, oils, gels, ointments, pastes, unguents, emulsionsand suspensions will contain an amount of digestive enzymes, andoptionally one or more other anti-infective agents, in a totalconcentration of between about 0.125 and about 10% by weight or more,recognizing again that optimal dosages may differ only by 0.05% byweight, so that representative cream and lotion embodiments will includeevery 0.05% by weight concentration increment within this range.

The topical compositions of the present invention are used to treat skininfections and wound infections such as surface wounds and penetratingwounds. Wounds suitable for treatment include wounds in skin abrasions,skin or surface cuts, decubiti, burns and surgical wounds. The topicalcompositions of the present invention can be used as well to decolonizepopulations of E. coli bacteria to prevent secondary, including thepre-treatment of areas prior to surgery or catheter insertion.

Mucosal delivery formulations can include digestive enzymes as describedherein combined or coordinately administered with a suitable carrier orvehicle for mucosal delivery. As used herein, the term “carrier” means apharmaceutically acceptable solid or liquid filler, diluent orencapsulating material. A water-containing liquid carrier can containpharmaceutically acceptable additives such as acidifying agents,alkalizing agents, antimicrobial preservatives, antioxidants, bufferingagents, chelating agents, complexing agents, solubilizing agents,humectants, solvents, suspending and/or viscosity-increasing agents,tonicity agents, wetting agents or other biocompatible materials. Atabulation of ingredients listed by the above categories, can be foundin the U.S. Pharmacopeia National Formulary, pp. 1857-1859, 1990. Someexamples of the materials which can serve as pharmaceutically acceptablecarriers are sugars, such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols such as glycerin, sorbitol, mannitoland polyethylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer'ssolution, ethyl alcohol and phosphate buffer solutions, as well as othernon toxic compatible substances used in pharmaceutical formulations.Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfateand magnesium stearate, as well as coloring agents, release agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions,according to the desires of the formulator. Examples of pharmaceuticallyacceptable antioxidants include water soluble antioxidants such asascorbic acid, cysteine hydrochloride, sodium bisulfite, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidantssuch as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and thelike; and metal-chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid andthe like. The amount of digestive enzymes that can be combined with thecarrier materials to produce a single dosage form will vary dependingupon the particular mode of administration.

Mucosal formulations are generally sterile, particulate free and stablefor pharmaceutical use. As used herein, the term “particulate free”means a formulation that meets the requirements of the USP specificationfor small volume parenteral solutions. The term “stable” means aformulation that fulfills all chemical and physical specifications withrespect to identity, strength, quality, and purity that have beenestablished according to the principles of Good Manufacturing Practice,as set forth by appropriate governmental regulatory bodies.

Within the mucosal delivery compositions, various delivery-enhancingagents can be employed which enhance delivery of digestive enzymes intoor across a mucosal surface. As used herein, “mucosal delivery-enhancingagents” include agents which enhance the release or solubility (e.g.,from a formulation delivery vehicle), diffusion rate, penetrationcapacity and timing, uptake, residence time, stability, effectivehalf-life, peak or sustained concentration levels, clearance and otherdesired mucosal delivery characteristics (e.g., as measured at the siteof delivery, or at a selected target site of activity such as thebloodstream or central nervous system) of digestive enzymes or otherbiologically active compound(s). Enhancement of mucosal delivery canthus occur by any of a variety of mechanisms, for example by increasingthe diffusion, transport, persistence or stability of the digestiveenzymes, increasing membrane fluidity, modulating the availability oraction of calcium and other ions that regulate intracellular orparacellular permeation, solubilizing mucosal membrane components (e.g.,lipids), changing non-protein and protein sulfhydryl levels in mucosaltissues, increasing water flux across the mucosal surface, modulatingepithelial junctional physiology, reducing the viscosity of mucusoverlying the mucosal epithelium, reducing mucociliary clearance rates,and other mechanisms.

While the mechanism of absorption promotion may vary with differentintranasal delivery-enhancing agents of the invention, useful reagentsin this context will not substantially adversely affect the mucosaltissue and will be selected according to the physicochemicalcharacteristics of the particular digestive enzymes or other active ordelivery-enhancing agent. In this context, delivery-enhancing agentsthat increase penetration or permeability of mucosal tissues will oftenresult in some alteration of the protective permeability barrier of themucosa. For such delivery-enhancing agents to be of value within theinvention, it is generally desired that any significant changes inpermeability of the mucosa be reversible within a time frame appropriateto the desired duration of drug delivery. Furthermore, there should beno substantial, cumulative toxicity, nor any permanent deleteriouschanges induced in the barrier properties of the mucosa with long-termuse.

In some embodiments, absorption-promoting agents for coordinateadministration or combinatorial formulation with the digestive enzymesas described herein are selected from small hydrophilic molecules,including but not limited to, dimethyl sulfoxide (DMSO),dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones.Alternatively, long-chain amphipathic molecules, for example,deacylmethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid, andthe bile salts, may be employed to enhance mucosal penetration of thedigestive enzymes. In additional aspects, surfactants (e.g.,polysorbates) are employed as adjunct compounds, processing agents, orformulation additives to enhance intranasal delivery of the digestiveenzymes. These penetration-enhancing agents typically interact at eitherthe polar head groups or the hydrophilic tail regions of molecules thatcomprise the lipid bilayer of epithelial cells lining the nasal mucosa(Barry, Pharmacology of the Skin, Vol. 1, pp. 121-137, Shroot et al.,Eds., Karger, Basel, 1987; and Barry, J. controlled Release 6:85-97,1987). Interaction at these sites may have the effect of disrupting thepacking of the lipid molecules, increasing the fluidity of the bilayer,and facilitating transport of the digestive enzymes across the mucosalbarrier. Interaction of these penetration enhancers with the polar headgroups may also cause or permit the hydrophilic regions of adjacentbilayers to take up more water and move apart, thus opening theparacellular pathway to transport of the digestive enzymes. In additionto these effects, certain enhancers may have direct effects on the bulkproperties of the aqueous regions of the nasal mucosa. Agents such asDMSO, polyethylene glycol, and ethanol can, if present in sufficientlyhigh concentrations in delivery environment (e.g., by pre-administrationor incorporation in a therapeutic formulation), enter the aqueous phaseof the mucosa and alter its solubilizing properties, thereby enhancingthe partitioning of the digestive enzymes from the vehicle into themucosa.

Additional mucosal delivery-enhancing agents that are useful within thecoordinate administration and processing methods and combinatorialformulations of the invention include, but are not limited to, mixedmicelles; enamines; nitric oxide donors (e.g.,S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4, which are preferablyco-administered with an NO scavenger such as carboxy-PITO or doclofenacsodium); sodium salicylate; glycerol esters of acetoacetic acid (e.g.,glyceryl-1,3-diacetoacetate or1,2-isopropylideneglycerine-3-acetoacetate); and other release-diffusionor intra- or trans-epithelial penetration-promoting agents that arephysiologically compatible for mucosal delivery. Otherabsorption-promoting agents are selected from a variety of carriers,bases and excipients that enhance mucosal delivery, stability, activityor trans-epithelial penetration of the digestive enzymes. These include,inter alia, cyclodextrins and β-cyclodextrin derivatives (e.g.,2-hydroxypropyl-β-cyclodextrin andheptakis(2,6-di-O-methyl-β-cyclodextrin). These compounds, optionallyconjugated with one or more of the active ingredients and furtheroptionally formulated in an oleaginous base, enhance bioavailability inthe mucosal formulations of the invention. Yet additionalabsorption-enhancing agents adapted for mucosal delivery includemedium-chain fatty acids, including mono- and diglycerides (e.g., sodiumcaprate-extracts of coconut oil, Capmul), and triglycerides (e.g.,amylodextrin, Estaram 299, Miglyol 810).

The mucosal therapeutic and prophylactic compositions may besupplemented with any suitable penetration-promoting agent thatfacilitates absorption, diffusion, or penetration of digestive enzymesacross mucosal barriers. The penetration promoter may be any promoterthat is pharmaceutically acceptable. Thus, in more detailed aspects ofthe invention compositions are provided that incorporate one or morepenetration-promoting agents selected from sodium salicylate andsalicylic acid derivatives (e.g., acetyl salicylate, choline salicylate,salicylamide); amino acids and salts thereof (e.g. monoaminocarboxlicacids such as glycine, alanine, phenylalanine, proline, hydroxyproline;hydroxyamino acids such as serine; acidic amino acids such as asparticacid, glutamic acid; and basic amino acids such as lysine—inclusive oftheir alkali metal or alkaline earth metal salts); and N-acetylaminoacids (N-acetylalanine, N-acetylphenylalanine, N-acetylserine,N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N-acetylproline,N-acetylhydroxyproline, etc.) and their salts (alkali metal salts andalkaline earth metal salts). Also provided as penetration-promotingagents within the methods and compositions of the invention aresubstances which are generally used as emulsifiers (e.g. sodium oleylphosphate, sodium lauryl phosphate, sodium lauryl sulfate, sodiummyristyl sulfate, polyoxyethylene alkyl ethers, polyoxyethylene alkylesters, etc.), caproic acid, lactic acid, malic acid and citric acid andalkali metal salts thereof, pyrrolidonecarboxylic acids,alkylpyrrolidonecarboxylic acid esters, N-alkylpyrrolidones, prolineacyl esters, and the like.

5. Compositions for Other Routes of Administration

Other routes of administration, such as transdermal patches, includingiontophoretic and electrophoretic devices, and rectal administration,are also contemplated herein.

Transdermal patches, including iotophoretic and electrophoretic devices,are well known to those of skill in the art. For example, such patchesare disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533,6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and5,860,957.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The weight of a rectal suppository, inone embodiment, is about 2 to 3 gm.

Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

6. Sustained Release Formulations

Also provided are sustained release formulations to deliver thedigestive enzymes to the desired target. It is understood that thedigestive enzymes levels are maintained over a certain period of time asis desired and can be easily determined by one skilled in the art. Suchsustained and/or timed release formulations may be made by sustainedrelease means of delivery devices that are well known to those ofordinary skill in the art, such as those described in U.S. Pat. Nos.3,845,770; 3,916,899; 3,536,809; 3, 598,123; 4,008,719; 4,710,384;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556 and 5,733,566, the disclosures of which are each incorporatedherein by reference. These pharmaceutical compositions can be used toprovide slow or sustained release of one or more digestive enzymesusing, for example, hydroxypropylmethyl cellulose, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or the like. Suitablesustained release formulations known to those skilled in the art,including those described herein, may be readily selected for use withthe pharmaceutical compositions provided herein. Thus, single unitdosage forms suitable for oral administration, such as, but not limitedto, tablets, capsules, gelcaps, caplets, powders and the like, that areadapted for sustained release are contemplated herein.

In one embodiment, the sustained release formulation contains an activecompound such as, but not limited to, microcrystalline cellulose,maltodextrin, ethylcellulose, and magnesium stearate. As describedabove, all known methods for encapsulation which are compatible withproperties of the disclosed digestive enzymes are contemplated herein.The sustained release formulation is encapsulated by coating particlesor granules of the pharmaceutical compositions provided herein withvarying thickness of slowly soluble polymers or by microencapsulation.In one embodiment, the sustained release formulation is encapsulatedwith a coating material of varying thickness (e.g. about 1 micron to 200microns) that allow the dissolution of the pharmaceutical compositionabout 48 hours to about 72 hours after administration to a mammal. Inanother embodiment, the coating material is a food-approved additive.

In another embodiment, the sustained release formulation is a matrixdissolution device that is prepared by compressing the drug with aslowly soluble polymer carrier into a tablet. In one embodiment, thecoated particles have a size range between about 0.1 to about 300microns, as disclosed in U.S. Pat. Nos. 4,710,384 and 5,354,556, whichare incorporated herein by reference in their entireties. Each of theparticles is in the form of a micromatrix, with the active ingredientuniformly distributed throughout the polymer.

The digestive enzymes provided herein can be formulated as a sustainedand/or timed release formulation. All sustained release pharmaceuticalproducts have a common goal of improving drug therapy over that achievedby their non-sustained counterparts. Ideally, the use of an optimallydesigned sustained release preparation in medical treatment ischaracterized by a minimum of digestive enzymes being employed to cureor control the condition. Advantages of sustained release formulationsmay include: 1) extended activity of the composition, 2) reduced dosagefrequency, and 3) increased patient compliance. In addition, sustainedrelease formulations can be used to affect the time of onset of actionor other characteristics, such as blood levels of the composition, andthus can affect the occurrence of side effects.

The sustained release formulations provided herein are designed toinitially release an amount of the therapeutic composition that promptlyproduces the desired therapeutic effect, and gradually and continuallyrelease of other amounts of compositions to maintain this level oftherapeutic effect over an extended period of time. In order to maintainthis constant level in the body, the therapeutic composition must bereleased from the dosage form at a rate that will replace thecomposition being metabolized and excreted from the body.

The sustained release of an active ingredient may be stimulated byvarious inducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds.

Preparations for oral administration may be suitably formulated to givecontrolled release of the digestive enzymes. In one embodiment, thedigestive enzymes are formulated as controlled release powders ofdiscrete microparticles that can be readily formulated in liquid form.The sustained release powder comprises particles containing an activeingredient and optionally, an excipient with at least one non-toxicpolymer.

The powder can be dispersed or suspended in a liquid vehicle and willmaintain its sustained release characteristics for a useful period oftime. These dispersions or suspensions have both chemical stability andstability in terms of dissolution rate. The powder may contain anexcipient comprising a polymer, which may be soluble, insoluble,permeable, impermeable, or biodegradable. The polymers may be polymersor copolymers. The polymer may be a natural or synthetic polymer.Natural polymers include polypeptides (e.g., zein), polysaccharides(e.g., cellulose), and alginic acid. Representative synthetic polymersinclude those described, but not limited to, those described in column3, lines 33-45 of U.S. Pat. No. 5,354,556, which is incorporated byreference in its entirety. Particularly suitable polymers include thosedescribed, but not limited to those described in column 3, line46—column 4, line 8 of U.S. Pat. No. 5,354,556 which is incorporated byreference in its entirety.

The sustained release compositions provided herein may be formulated forparenteral administration, e.g., by intramuscular injections or implantsfor subcutaneous tissues and various body cavities and transdermaldevices. In one embodiment, intramuscular injections are formulated asaqueous or oil suspensions. In an aqueous suspension, the sustainedrelease effect is due to, in part, a reduction in solubility of thedigestive enzymes upon complexation or a decrease in dissolution rate. Asimilar approach is taken with oil suspensions and solutions, whereinthe release rate of digestive enzymes is determined by partitioning ofthe digestive enzymes out of the oil into the surrounding aqueousmedium. Only digestive enzymes which are oil soluble and have thedesired partition characteristics are suitable. Oils that may be usedfor intramuscular injection include, but are not limited to, sesame,olive, arachis, maize, almond, soybean, cottonseed and castor oil.

Coadministration with Other Pharmaceutical Compositions

The pharmaceutical compositions can be used on their own, and/or incombination with other therapeutic or antibiotic (e.g., anti-E. coli)regimens. For example, a patient can be administered other therapeuticagents, such as anti-inflammatories or anesthetics, to address otheraspects of an E. coli infection (e.g., pain, tissue damage) or stillother illnesses that the patient may be facing. In other embodiments, apatient can be administered a pharmaceutical composition as describedherein, and one or more additional antibiotics. The one or moreadditional antibiotics can be effective against E. coli or otherbacteria, or both (e.g., if the patient has multiple infections) and canbe in the same or a different format from the present pharmaceuticalcompositions (e.g., one can be a liquid and one can be a topicalantibiotic). The major classes of antibiotics are (1) the β-lactams,including the penicillins, cephalosporins and monobactams; (2) theaminoglycosides, e.g., gentamicin, tobramycin, netilmycin, and amikacin;(3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) thefluoroquinolones, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6)vancomycin; (7) the macrolides, which include for example, erythromycin,azithromycin, and clarithromycin; and (8) other antibiotics, e.g., thepolymyxins, chloramphenicol and the lincosamides.

In some embodiments, the additional antibiotic can be a beta-lactamantibiotic (e.g., a penicillin or penicillin derivatives, acephalosporin, a monobactam, a penam, a penem, an oxapenam, a carbapenemor a cabapenam, a cephem, a carbacephem, an oxacephem, a monobactam). Insome embodiments, the additional antibiotic can be a beta-lactamaseinhibitor. In some embodiments, the additional antibiotic can be anaminoglycosidic antibiotic. In some embodiments, the additionalantibiotic is selected from penicillin or a penicillin derivative,oxacillin, amoxicillin, nafcillin, cloxacillin, methicillin, temocillin,ampicillin, co-amoxiclav, azlocillin, carbenicillin, ticarcillin,mezlocillin, piperacillin, cephalexin, cephalothin, cefazolin, cefaclor,cefuroxime, cefamandole, cefotetan, cefoxitin, ceftriaxone, cefotaxime,cefpodoxime, ceftazidime, cefepime, cefpirome, vancomycin, teicoplanin,telavancin, bleomycin, ramoplanin, decaplanin, oritavancin, anddalbavancin.

An antibiotic composition described herein and an additional antibioticcan be administered separately or in a single dosage form. Ifseparately, they can be administered in any order and relativefrequency.

Disinfectants and Sanitizers

Compositions comprising one or more digestive enzymes as describedherein can also be used as disinfectants and sanitizers, e.g., todisinfect inanimate objects and surfaces in, without limitation,hospital, health care, home, and community settings by eradicating,attenuating or reducing E. coli in such locations. Disinfectants areantimicrobial agents that are applied to non-living objects to destroymicroorganisms. Disinfectants should generally be distinguished fromantibiotics that destroy microorganisms within the body, and fromantiseptics, which destroy microorganisms on living tissue. Sanitizersare disinfectants that reduce the number of microorganisms to a safelevel. One definition of a sanitizer states that a sanitizer must becapable of killing 99.999%, known as a 5 log reduction, of a specificbacterial test population, and to do so within 30 seconds. The maindifference between a sanitizer and a disinfectant is that at a specifieduse dilution, the disinfectant must have a higher kill capability forpathogenic bacteria compared to that of a sanitizer.

A disinfectant or sanitizer as described herein can include one or moredigestive enzymes, in various embodiments as described previously above,and can optionally include other active and inactive ingredients,including stabilizers (e.g., enzyme stabilizers), other disinfectantsknown to those having ordinary skill in the art, formulation excipients,colorants, perfumes, etc. One having ordinary skill in the art canselect the additional active or inactive ingredients to include in adisinfectant. Examples of additional disinfectants include: sources ofactive chlorine (i.e., hypochlorites, chloramines, dichloroisocyanurateand trichloroisocyanurate, wet chlorine, chlorine dioxide etc.); sourcesof active oxygen (peroxides, such as peracetic acid, potassiumpersulfate, sodium perborate, sodium percarbonate and urea perhydrate);iodine and iodophor solutions (iodpovidone (povidone-iodine, Betadine),Lugol's solution, iodine tincture, iodinated nonionic surfactants);concentrated alcohols (mainly ethanol, 1-propanol, called alson-propanol and 2-propanol, called isopropanol and mixtures thereof;further, 2-phenoxyethanol and 1- and 2-phenoxypropanols); phenolicsubstances (such as phenol (also called “carbolic acid”), cresols(called “Lysole” in combination with liquid potassium soaps),halogenated (chlorinated, brominated) phenols, such as hexachlorophene,triclosan, trichlorophenol, tribromophenol, pentachlorophenol, Dibromoland salts thereof); cationic surfactants, such as some quaternaryammonium cations (such as benzalkonium chloride, cetyl trimethylammoniumbromide or chloride, didecyldimethylammonium chloride, cetylpyridiniumchloride, benzethonium chloride) and others; non-quarternary compounds,such as chlorhexidine, glucoprotamine, octenidine dihydrochloride;strong oxidizers, such as ozone and permanganate solutions; heavy metalsand their salts, such as colloidal silver, silver nitrate, mercurychloride, phenylmercury salts, copper sulfate, and copperoxide-chloride; concentrated strong acids (phosphoric, nitric, sulfuric,amidosulfuric, toluenesulfonic acids) and alkalis (sodium, potassium,calcium hydroxides), such as of pH<1 or >13, particularly under elevatedtemperature (above 60° C.). In some cases, a disinfectant as describedherein will consist essentially of the one or more digestive enzymes. Insome cases, a disinfectant will consist essentially of the one or moredigestive enzymes, and will not include additional disinfecting agents.

A disinfectant composition comprising one or more digestive enzymes asdescribed herein can be incorporated with other ingredients to form avariety of disinfectant products including but not limited to handcleansers, mouthwashes, surgical scrubs, body splashes, hand sanitizergels and foams, disinfectant wipes, and similar personal care products.Additional types of products include disinfectant foams, creams,mousses, and the like, and compositions containing organic and inorganicfiller materials, such as emulsions, lotions, creams, pastes, and thelike. The compositions can also be used as an antibacterial cleanser forhard surfaces, for example, sinks and countertops in hospitals, foodservice areas, and meat processing plants. The disinfectant compositionscan also be used as disinfectant fogs and disinfectant mists. Thepresent digestive enzyme compositions can be manufactured as diluteready-to-use compositions, or as concentrates that are diluted prior touse. The various products in which the disinfectants are used may alsoinclude fragrances, depending on the nature of the product. For example,a pine or lemon fragrance may be desirable for use for kitchen cleaningwipes because of their appealing association with cleanliness to manyconsumers. Further, gels or aerosols may also be fragranced for similaror other reasons.

In one embodiment, the disinfectant compositions can be used to makedisinfectant wipes. A disinfectant wipe can be used to clean a varietyof hard and other surfaces, including, for example, human hands andskin, medical instruments and devices, countertops, floors, walls, andwindows. Wipes can be made of a variety of fabrics. Fabrics are definedto include cloths and papers, as well as woven and non-woven materials.The woven or nonwoven fabrics can be made of suitable materials such asrayon, nylon, or cotton, and combinations thereof. Examples of nonwovenfabrics are described in U.S. Pat. Nos. 3,786,615; 4,395,454; and4,199,322; which are hereby incorporated by reference. The fabrics orpapers can be impregnated with the disinfectant solution by any methodknown in the art. The wipes can be packaged in any manner known in theart including individual blister-packs or wrapped or stackedmulti-packs.

In another embodiment, the disinfectant composition comprising one ormore digestive enzymes can be formulated into a gel or gelatinoussanitization composition. In addition to the disinfectant compositions,the gel sanitizers can include a thickening or gelling agent, wherein“thickening agent” and “gelling agent” are used interchangeably. As usedherein, the terms “gel” or “gelatinous” sanitization compositions refersto a disinfectant liquid substances that can have a viscosity from about1,000 centipoise to about 100,000 centipoise, or from 2,000 centipoiseto 50,000 centipoise in another embodiment, though these ranges are notintended to be limiting. For example, a hand gel may be considerablyless viscous than a gel used for industrial cleaning or disinfectantpurposes. Examples of gelling or thickening agents include but are notlimited to natural gum such as guar and guar derivatives, a syntheticpolymer, a clay, an oil, a wax, aloe vera gel, an acrylate homopolymer,an acrylate copolymer, a carbomer, cellulose, a cellulose derivative,algin, an algin derivative, a water-insoluble C8-C20 alcohol,carrageenan, fumed silica, mixtures thereof, and the like. The gellingagent can be present in the gelatinous sanitation composition in anamount from about 0.1 wt % to 50 wt % of the gelatinous composition. Inanother embodiment, the gelling agent is present in an amount from 0.25wt % to 10 wt % of the gelatinous composition. The amount of gellingagent can be dependent on a variety of factors including the type ofgelling agent and the desired viscosity of the gel. The gelatinoussanitizers can be used for a variety of applications includingsanitization of human skin e.g., gel hand sanitizer, and hard surfacesanitation. In one particular embodiment, the disinfectant compositioncan be mixed with natural aloe gel to form a disinfectant aloeformulation. Such a formulation would be useful for application toburns, skin infections, and other irritations. The aloe may act as athickening agent, or may also include another thickening or gellingagent as described above, depending on the desired viscosity of thedisinfectant gel.

In another embodiment, a disinfectant composition comprising one or moredigestive enzymes can be formulated into a disinfectant foam or foamingcomposition. The disinfectant foams or foaming compositions include thedisinfectant composition and foaming agents. Any foaming agent known inthe art can be used depending on the desired application andcharacteristics of the resulting disinfectant foam. As with thedisinfectant composition, the disinfectant foams of the presentinvention can be used in both human (e.g. hand washing) and industrialapplications.

In another embodiment, the disinfectant composition comprising one ormore digestive enzymes can be in the form of a disinfectant aerosol orfog. Fogging, also referred to as thermal fogging, is the process bywhich disinfectants are aerosolized. The aerosol particles of thedisinfectant are suspended within the air for a period of time in orderto disinfect both the air itself and surfaces, including inaccessibleparts of a structure such as air vents. The aerosolized particles ofdisinfectant can have a particle size of from about 5 μm to about 200μm. In another embodiment, the aerosolized particle can have a particlesize of from about 20 μm to about 150 μm.

Methods for evaluating the disinfectant ability of a particularcomposition are known to those having ordinary skill in the art.Typically, the relative effectiveness of a disinfectant can be measuredby comparing how well it disinfects as compared to a known disinfectant.Phenol is one known disinfectant standard, and the corresponding ratingsystem is called the “Phenol coefficient”. The disinfectant to be testedis compared with phenol on a standard microbe, e.g., E. coli or S.aureus. Disinfectants that are more effective than phenol have acoefficient>1. Those that are less effective have a coefficient<1. Tocalculate phenol coefficient, the concentration of the test compound atwhich the compound kills the test organism in 10 minutes, but not in 5minutes, is divided by the concentration of phenol that kills theorganism under the same conditions. The phenol coefficient may bedetermined in the presence of a standard amount of added organic matteror in the absence of organic matter. One particular phenol coefficientassay uses the Rideal-Walker method. The U.S. Department of Agriculturealso has a method that gives a U.S. Department of Agriculturecoefficient. Other methods are known to those having ordinary skill inthe art.

A disinfectant as described herein can have a phenol coefficient for S.aureus that is >1, e.g., greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5,1.75, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 12, 14, 16,18, or higher. In some cases, the phenol coefficient is in the rangefrom about 2 to about 20, e.g., about 4 to about 10, about 2 to about 6,about 6 to about 12, or about 10 to about 15.

A disinfectant as described herein can have a phenol coefficient for E.coli that is >1, e.g., greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75,2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18.In some cases, the phenol coefficient is in the range from about 2 toabout 20, e.g., about 4 to about 10, about 2 to about 6, about 6 toabout 12, or about 10 to about 15.

A disinfectant or sanitizer as described herein can be bacteriocidaland/or bacteriostatic to E. coli. In some embodiments, a disinfectant orsanitizer as described herein can be bacteriocidal and/or bacteriostaticagainst STEC, ETEC, or EPEC, or any 2, or all 3.

Detergents

A disinfectant composition comprising one or more digestive enzymesdescribed herein can also be formulated as a detergent. A detergent is amaterial intended to assist cleaning A detergent can contain one or moredigestive enzymes, as described previously, in a formulation suitable tomaintain its disinfectant ability, and can contain optional active orinactive ingredients, e.g., enzyme stabilizers, additionaldisinfectants, bleaches, soaps, surfactants, colorants and perfumes,abrasives, pH modifiers, acids, alkalis, or caustic compounds, watersofteners, oxidants, suspending agents, fabric softeners, foaming agentsor anti-foaming agents, viscosity modifiers, corrosion inhibitors, andoptical brighteners. A detergent as described herein can bebacteriostatic and/or bacteriocidal to E. coli, and in some embodimentscan be bacteriostatic and/or bacteriocidal against STEC, ETEC, or EPEC,or any 2, or all 3.

A detergent composition comprising one or more digestive enzymes,especially those made for use with water, can include additionalcomponents such as surfactants to ‘cut’ (dissolve) grease and to wetsurfaces, abrasives to scour, substances to modify pH or to affectperformance or stability, acids for descaling or caustics to break downorganic compounds, water softeners to counteract the effect of“hardness” ions, oxidants (oxidizers) for bleaching, disinfection, andbreaking down organic compounds, non-surfactant materials that keep dirtin suspension, enzymes to digest proteins, fats, or carbohydrates instains or to modify fabric feel, ingredients that modify the foamingproperties of the cleaning surfactants, to either stabilize orcounteract foam, ingredients to increase or decrease the viscosity ofthe solution, or to keep other ingredients in solution, in a detergentsupplied as a water solution or gel, ingredients that affect aestheticproperties of the item to be cleaned, or of the detergent itself beforeor during use, such as optical brighteners, fabric softeners, colors,and perfumes, ingredients such as corrosion inhibitors to counteractdamage to equipment with which the detergent is used, ingredients toreduce harm or produce benefits to skin, when the detergent is used bybare hand on inanimate objects or used to clean skin, and preservativesto prevent spoilage of other ingredients.

The detergent composition may be in any convenient dry form, e.g., abar, a tablet, a powder, a granule or a paste. It may also be a liquiddetergent.

The digestive enzyme(s) of the detergent composition of the inventionmay be stabilized using conventional stabilizing agents, e.g., a polyolsuch as propylene glycol or glycerol, a sugar or sugar alcohol, lacticacid, boric acid, or a boric acid derivative, e.g., an aromatic borateester, or a phenyl boronic acid derivative such as 4-formylphenylboronic acid, and the composition may be formulated as described in e.g.WO 92/19709 and WO 92/19708.

Antiseptics

Various embodiments of compositions comprising on or more digestiveenzymes can also be used as antiseptic agents, e.g., to reduce,eradicate, or attenuate E. coli on skin or other living tissues.Antiseptics are antimicrobial substances that are applied to livingtissue/skin to reduce the possibility of infection, sepsis, orputrefaction. They should generally be distinguished from antibioticsthat destroy bacteria within the body, and from disinfectants, whichdestroy microorganisms found on non-living objects. Some antiseptics aretrue germicides, capable of destroying microbes (bacteriocidal), whileothers are bacteriostatic and only prevent or inhibit their growth.

The presently described antiseptics can find particular use in hospitalor healthcare settings, e.g., in hand, facial or body-wash formulations;as antiseptics for use prior to and post surgical treatment; and asantiseptics for use in cleansing and treating wounds, such as traumaticor surgical wounds. In the community setting, antiseptics are useful inany setting where community-acquired infections are of concern, e.g.,daycare settings, large institutions, schools, etc. Antiseptics can alsobe useful in the home setting in hand, facial or body-wash formulations,or for treating wounds.

An antiseptic can include one or more digestive enzymes, in variousembodiments as described previously, and can optionally include one ormore active or inactive ingredients, such as other antiseptic agentsknown to those having ordinary skill in the art, stabilizers (e.g.,enzyme stabilizers), colorants, perfumes, and other excipients. Examplesof antiseptic agents to include with the one or more digestive enzymesinclude alcohols (e.g., ethanol, 1- and 2-propanol, or mixturesthereof), quaternary ammonium compounds (benzalkonium chloride, cetyltrimethylammonium bromide, cetylpyridinium chloride, and benzethoniumchloride), boric acid, chlorhexidine gluconate, peroxides (e.g.,hydrogen peroxide, benzoyl peroxide); iodine and iodophor solutions(e.g., povidone-iodine), octenidine dihydrochloride, Phenolic (carbolicacid) and phenolic derivative compounds, sodium chloride, sodiumhypochlorite, and calcium hypochlorite.

An antiseptic as described herein can be bacteriocidal and/orbacteriostatic to E. coli, and in some embodiments can be bacteriocidaland/or bacteriostatic to STEC, ETEC, or EPEC, or any 2, or all 3.

In one embodiment, an antiseptic composition comprises one or moredigestive enzymes and optionally one or more of an anti-inflammatoryagent, an analgesic, or an anaesthetic.

An anti-inflammatory agents can include steroidal and non-steroidalanti-inflammatory compounds. In one embodiment, the antisepticcomposition includes one or more steroids. In one embodiment, theantiseptic compositions can comprise an anti-inflammatory agent that isa non-steroidal anti-inflammatory drug. Non-limiting examples ofsuitable non-steroidal anti-inflammatory drugs include aspirin (Anacin,Ascriptin, Bayer, Bufferin, Ecotrin, Excedrin), choline and magnesiumsalicylates (CMT, Tricosal, Trilisate), choline salicylate (Artliropan),celecoxib (Celebrex), diclofenac potassium (Cataflam), diclofenac sodium(Voltaren, Voltaren XR), diclofenac sodium with misoprostol (Arthrotec),difiunisal (Dolobid), etodolac (Lodine, Lodine XL), fenoprofen calcium(Nalfon), flurbiprofen (Ansaid), ibuprofen (Advil, Motrin, Motrin IB,Nuprin), indomethacin (Indocin, Indocin SR), ketoprofen (Actron, Orudis,Orudis KT, Oruvail), magnesium salicylate (Arthritab, Bayer Select,Doan's Pills, Magan, Mobidin, Mobogesic), meclofenamate sodium(Meclomen), mefenamic acid (Ponstel), meloxicam (Mobic), nabumetone(Relafen), naproxen (Naprosyn, Naprelan), naproxen sodium (Aleve,Anaprox), oxaprozin (Daypro), piroxicam (Feldene), rofecoxib (Vioxx),salsalate (Amigesic, Anaflex 750, Disalcid, Marthritic, Mono-Gesic,Salflex, Salsitab), sodium salicylate, sulindac (Clinoril), tolmetinsodium (Tolectin), valdecoxib (Bextra), or a combination thereof.

Non-limiting examples of analgesics include acetylsalicylic acid,codeine, ibuprofen, acetaminophen, or tea tree oil. Non-limitingexamples of anaesthetics include xylocaine, prilocaine or benzocaine.

Exemplary methods of testing candidate antiseptic compositions areprovided below. One skilled in the art will understand that othermethods of testing the antiseptic compositions are known in the art andare also suitable for testing candidate antiseptic compositions.

In vitro methods of determining the ability of candidate antisepticcompositions to kill or inhibit the growth of microbial cells such as E.coli are well-known in the art. In general, these methods involvecontacting a culture of the cells of interest with variousconcentrations of the candidate antiseptic compositions and monitoringthe growth of the cell culture relative to an untreated control culture.A second control culture comprising cells contacted with a knownanti-microbial agent may also be included in such tests, if desired.

For example, the ability of a candidate antiseptic composition toinhibit the growth of microbial cells can readily be determined bymeasurement of the minimum inhibitory concentration (MIC) for theantiseptic composition. The MIC is defined as the lowest concentrationthat inhibits growth of the organism to a pre-determined extent. Forexample, a MIC₁₀₀ value is defined as the lowest concentration thatcompletely inhibits growth of the organism, whereas a MIC₉₀ value isdefined as the lowest concentration that inhibits growth by 90% and aMIC₅₀ value is defined as the lowest concentration that inhibits growthby 50%. MIC values are sometimes expressed as ranges, for example, theMIC₁₀₀ for an antiseptic composition may be expressed as theconcentration at which no growth is observed or as a range between theconcentration at which no growth is observed and the concentration ofthe dilution which immediately follows.

Anti-bacterial MICs for candidate antiseptic compositions can bemeasured using a broth macro- or microdilution assay (see Amsterdam, D.(1996) “Susceptibility testing of antimicrobials in liquid media,” pp.52-111. In Loman, V., ed. Antibiotics in Laboratory Medicine, 4th ed.Williams and Wilkins, Baltimore, Md.). A standardised anti-bacterialsusceptibility test is provided by the National Committee for ClinicalLaboratory Standards (NCCLS) as NCCLS, 2000; document M7-A58.

In the classical broth microdilution method, the candidate antisepticcomposition is diluted in culture medium in a sterile, covered 96-wellmicrotiter plate. An overnight culture of a single bacterial colony isdiluted in sterile medium such that, after inoculation, each well in themicrotiter plate contains an appropriate number of colony forming units(CFU) AnI (typically, approximately 5×105 CFU/ml). Culture medium only(containing no bacteria) is also included as a negative control for eachplate and known antibiotics are often included as positive controls. Theinoculated microtiter plate is subsequently incubated at an appropriatetemperature (for example, 35° C.-37° C. for 16-48 hours). The turbidityof each well is then determined by visual inspection and/or by measuringthe absorbance, or optical density (OD), at 595 nm or 600 nm using amicroplate reader and is used as an indication of the extent ofbacterial growth.

Anti-microbial effects may also be expressed as the percentage (%)inhibition of growth of a given micro-organism over a pre-determinedperiod of time by treatment with a single concentration of a candidateantiseptic composition. This method provides a rapid method of assessingthe ability of an antiseptic composition to inhibit microbial growth,for example, prior to conducting more in-depth tests, such as MICdeterminations or in vivo testing.

The ability of any of the present disinfectant, detergent, santitizer,and antiseptic compositions to kill or inhibit the growth of E. colibacteria can be tested using methods well-known in the art, includingthe various methods described above. Methods and protocols for testingcompositions against specific bacteria can be found, for example, inOfficial Methods of Analysis of the AOAC, 15th Ed., Arlington Va. 22201,USA (Association of Official Analytical Chemists (AOAC), Inc. 1990),Designation: E 1054-91 “Practices for Evaluation Inactivators ofAntimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, orPreserved Products” (American Society for Testing and Materials (ASTM),1991). As is also known in the art, in vitro Time-Kill evaluations canbe performed using a modification of the methods described in the DraftEuropean Standard, prEN 12054, “Chemical Disinfectants andAntiseptics—Products for Hygienic and Surgical Handrub andHandwash—Bactericidal Activity—Test Method and Requirements (1995).”Additional methods that may be used include the log reduction test,proliferation testing, the AOAC use dilution test, or the zone ofinhibition test. Other methods are described in the Examples below.

Kits

Also provided herein are kits. Typically, a kit includes one or morecompositions as described herein. In certain embodiments, a kit caninclude one or more delivery or administration systems, e.g., fordelivering or administering a composition as provided above, and/ordirections for use of the kit (e.g., instructions for treating apatient; instructions for disinfecting a surface). In anotherembodiment, the kit can include a composition as described herein and alabel, e.g., a label that indicates that the contents are to beadministered to a patient with a E. coli infection, or a label as to howto use the composition as a disinfectant, sanitizer, detergent, orantiseptic.

Methods of Use

Pharmaceutical compositions (e.g., antibiotic compositions) describedpreviously can be used to treat or prevent E. coli infections inanimals, e.g., mammals and birds. In particular, the pharmaceuticalcompositions can be used to ameliorate one or more symptoms and sideeffects of such infections and/or to reduce or eradicate the E. colibacterium causing the infection. The pharmaceutical compositions can bein any appropriate dosage form, as described previously. In certainembodiments, the antibiotic compositions described herein are used totreat wounds or lesions that have become infected, e.g., woundsresulting from trauma or surgery. Such use can reduce scarring andpromote wound healing in patients having infected wounds. Compositionsformulated for pharmaceutical use can also be employed prophylactically,e.g., as antiseptics. Such compositions find particular use in theprophylactic treatment of surgical incisions and other wounds, toprevent E. coli infection.

The digestive enzymes provided herein can be used to treat a variety ofdiseases and disorders associated with infection by E. coli bacteria orin which E. coli bacteria are implicated. Certain embodiments includeinfections associated with medical devices or prostheses, e.g. catheter,grafts, prosthetic heart valves, artificial joints, etc. In someembodiments, a composition comprising one or more digestive enzymes canbe coated onto the medical device either at manufacture of the device orafter manufacture but prior to insertion of the device.

Surgical wounds, especially those associated with foreign material, e.g.sutures may also be treated with the compositions provided herein. Asmany as 71% of all nosocomial infections occur in surgical patients, 40%of which are infections at the operative site. Despite efforts toprevent infection, it is estimated that between 500,000 and 920,000surgical wound infections complicate the approximately 23 millionsurgical procedures performed annually in the United States.

The digestive enzymes alone or with an antibiotic, anesthetic, oranti-inflammatory may be applied as an ointment, cream or liquid to thewound site or as a liquid in the wound prior to and during closure ofthe wound. Following closure, a composition comprising one or moredigestive enzymes could be applied at dressing changes. For wounds thatare infected, the composition could be applied topically and/orsystemically.

In early acute onset of osteomyelitis the vascular supply to the bone iscompromised by infection extending into surrounding tissue. Within thisnecrotic and ischemic tissue, the bacteria may be difficult to eradicateeven after an intense host response, surgery, and/or antibiotic therapy.The main organisms responsible are SA and E. coli.

The digestive enzymes could be administered systemically alone or incombination with other antibiotics. Treatment could be 2-6 weeks induration. The antibiotic could be given as a continuous infusion ormultiple administration during the day. A composition comprising one ormore digestive enzymes could be used as an antibiotic-impregnated cementor as antibiotic coated beads for joint replacement procedures.

Treatment or prevention of sepsis in immunocompromised host is alsoprovided. Treatment of infections in patients who are immunocompromisedby virtue of chemotherapy-induced granulocytopenia and immunosuppressionrelated to organ or bone marrow transplantation represents a significantchallenge. The neutropenic patient is especially susceptible tobacterial infection, so antibiotic therapy should be initiated promptlyto cover likely pathogens, if infection is suspected. Organisms likelyto cause infections in granulocytopenic patients are: SA and E. coli.

The digestive enzyme composition alone or with an antibiotic ispreferably administered orally or systemically for 2-6 weeks induration. The digestive enzymes could be given as a continuous infusionor via multiple administrations during the day.

Disinfectant, sanitizing, and detergent compositions as described hereincan be applied to non-living surfaces in the appropriate amounts andmanner to reduce or eradicate E. coli on such surfaces, and thus canreduce or prevent E. coli transmission and/or infectivity.Concentrations, timing, and frequency of treatment are parameters thatcan be determined by one having ordinary skill in the art.

Any surface can be disinfected with the described compositions,including a variety of medical devices used in the hospital orhealth-care setting. As used herein, “medical device” refers to anydevice for use in or on a patient, such as an implant or prosthesis.Such devices include, without limitation, synthetic vascular grafts,blood monitoring devices, artificial heart valves, scalpel, knife,scissors, spatula, expander, clip, tweezer, speculum, retractor, suture,surgical mesh, chisel, drill, level, rasp, saw, splint, caliper, clamp,forceps, hook, lancet, needle, cannula, curette, depressor, dilator,elevator, articulator, extractor, probe, staple, valve, catheter, stent,tubing, bowl, tray, sponge, snare, spoon, syringe, pacemaker, screw,plate, or pin.

Other community and hospital or health-care surfaces suspected ofharboring E. coli can be disinfected, including large or small surfaces(floors, tables, changing tables, beds, ventilation systems, tubs, doorhandles, counters, food service surfaces, etc.). The compositions canalso find use in hand or body washes, e.g., at points of entry tocommunity settings, hospital rooms, or bathrooms.

The compositions provided herein may be used in the manner of commondisinfectants or in any situation in which microorganisms areundesirable. For example, they may be used as surface disinfectants, incoatings for medical devices, in coatings for clothing, such as toinhibit growth of bacteria or repel mosquitoes, in filters for airpurification, such as on an airplane or in community or hospitalsettings, in water purification system, as constituents of shampoos andsoaps, as food preservatives, cosmetic preservatives, mediapreservatives, in herbicides or insecticides, as constituents ofbuilding materials, such as in silicone sealant, and in animal productprocessing, such as the curing of animal hides or in slaughterhouses.

For these purposes, typically the digestive enzymes alone or inconjunction with other disinfectants or detergents are included in thecompositions and applied with an appropriate applicator. They also maybe incorporated or impregnated into the material during manufacture,such as for an air filter, or otherwise applied to the material or theobject.

For example, in some embodiments, a composition described herein can bemixed in with the material, for example during manufacturing of thematerial or at a subsequent time. In addition, a composition can beapplied to the surface of a material, either during manufacturing or ata subsequent time. As used herein, the term “suitable material” meansany material on, to, or in which the digestive enzymes can be applied orincorporated, thereby incorporating an antimicrobial activity in/on thematerial. For example, a gauze pad on a bandage can be manufactured witha composition comprising one or more digestive enzymes in or on thegauze, and/or an ointment comprising one or more digestive enzymes canbe applied to the gauze thereby incorporating antimicrobial activity tothe gauze. Examples of suitable materials in which digestive enzymes maybe used, include, but are not limited to: foods, liquids, a medicaldevice (e.g. surgical instruments), a bead, a film, a monofilament, anunwoven fabric, sponge, cloth, a knitted fabric, a short fiber, a tube,a hollow fiber, an artificial organ, a catheter, a suture, a membrane, abandage, and gauze. The digestive enzymes may be applied or mixed intonumerous other types of materials that are suitable for use in medical,health, food safety, or environmental cleaning activities.

Veterinary Applications

The compositions described herein, in pharmaceutical ordisinfectant/sanitizer, detergent or antiseptic formats, can also beused in a variety of veterinary applications. For example, many mammals,including dogs, cats, and cows, can be infected with E. coli or act ascarriers of the bacteria. Accordingly, the present compositions can beused to treat animals infected with or suspected to carry E. coli inorder to treat the infection or to prevent transmission to otheranimals, including humans. Disinfectant and detergent compositions canbe used to treat animal living quarters and equipment that comes intocontact with the animals, while antiseptics and antibiotic formulationscan be used to treat animals to prevent or treat infection.

For example, the digestive enzymes provided herein can be used for theprevention and treatment of mastitis, particularly mastitis in dairycattle, though any mastitis can be treated using the digestive enzymesprovided herein. Mastitis in dairy cattle is an inflammation of themammary gland in response to intramammary bacterial infection,mechanical trauma, or chemical trauma. It is thought that contagiousmastitis is primarily caused by S. aureus and Streptococcal agalactiae.Environmental mastitis can be caused by a variety of different bacteria,including, but not limited to, K. pneumoniae, E. coli, Klebsiellaoxytoca, Enterobacter aerogenes, Streptococcal uberis, Streptococcalbovis, and Streptococcal dysgalactia.

In some embodiments, prevention of bovine mastitis can include dailyteat-dipping with a solution comprising one or more digestive enzymes.In some embodiments, the solution comprising one or more digestiveenzymes may further include one or more additional antibiotics. Wheninfection does occur, intramammary infusion of one or more digestiveenzymes may be implemented. As above, additional antibiotics may also beadministered in conjunction with the digestive enzymes. Typically, thedigestive enzymes are administered by intramammary injection; however,effective dosages may be administered parenterally, percutaneously, byimplant and also by dipping. In some embodiments, bovine mastitis can betreated by administering an effective amount of one or more digestiveenzymes to a cow. The administration may be a prophylacticadministration, in that all cattle in the herd are treated with adigestive enzyme composition, or the administration may occur wheninfection occurs in individual cows.

Introduction of S. aureus (also referred to as SA herein) bacteria andE. coli bacteria can occur during the preparation of beef, poultry, fishand pork products. Accordingly, in some embodiments, reduction ofinfection can be provided through administration of one or moredigestive enzymes to an animal (e.g., cow, chicken, turkey, fish, orpig) to reduce the presence of E. coli or SA bacteria in the intestinesof the animal. Administration may occur through any available methodincluding injection and through the introduction of one or moredigestive enzymes in feed.

In some embodiments, administration of one or more digestive enzymes toan animal may be used to prevent or reduce transmission of SA bacteriaor E. coli from the animal to other animals or humans. Administration ofthe digestive enzymes may be accomplished through any available methodknown in the art.

Food Applications

Also provided herein is a method of preventing SA or E. coli infectionof beef, poultry, fish, and pork. Beef processing is a common point ofcontamination: during the slaughtering process, the contents ofintestines or fecal material on the hide could mix with the meat, thusallowing bacteria to flourish in the warm, damp conditions. If theinfected parts are then ground, the bacteria go from the surface of thecut to the interior of the ground mass. Additionally, in the productionof ground beef, meat from multiple cattle is often ground together,enabling contamination from a single animal to infect an entire lot ofground beef Accordingly, in some embodiments, reduction of infection canbe provided through administration of one or more digestive enzymes to acow to reduce the presence of bacteria in the intestines of the cow.Administration may occur through any available method includinginjection and through the introduction of one or more digestive enzymesin feed. In another embodiment, the reduction of contamination of meatduring slaughter and grinding can be provided through the use of sprayscontaining one or more digestive enzymes as provided herein. Such spraysmay be used, for example, in the disinfection of slaughter and grindinginstruments or in the disinfection of the ground meat itself. Themethods described above can further be used during the slaughter andpreparation of poultry, fish and pork products (e.g., throughadministration or one or more digestive enzymes to poultry, fish and/orpigs prior to slaughter).

E. coli and SA bacteria can also be spread through unwashed fruits andvegetables. Accordingly, also provided herein is a method of washingfresh fruits and vegetables using a solution, wash, aerosol, fog, gel,or powder comprising one or more digestive enzymes as provided herein. Aproduce wash is a solution used to bathe the surface of produce, andtypically is in contact with the produce from about 30 sec to about 5min. A produce soak is a solution in which produce items are immersedfor a time from about 30 sec to about 30 min. However, the terms andsolutions can be used interchangeably unless otherwise distinguished. Itis understood that the temperature at which produce is washed or soakedwill influence the length of time necessary to reduce or inactivatebacteria thereon, with warmer temperatures leading to a shorter timenecessary for treatment.

The wash or soak solutions described herein can be used to reducebacterial number, especially bacterial pathogen number, on the surfacesof fruits, vegetables, raw cut meat products, fish, shellfish, at theconsumer level (in the household), in commercial food preparationenvironments, on fruits and/or vegetables prior to juicing, by wholesaleor retail operators, and/or at the level of the harvest, meat packingplant or slaughterhouse, fishing boat, and so on, without limitation.The present methods are particularly useful for inactivating E. coli onthe surfaces of fresh fruits and vegetables.

Methods for Evaluating Activity

Compositions described herein can be evaluated for a variety ofactivities by methods known to those having ordinary skill in the art.For example, enzymatic activities can be evaluated using standard enzymeassays. Minimum Inhibitory Concentrations (MIC) of the compositions canalso be evaluated by methods known to those having ordinary skill in theart, as described above. Other assays, including the Phenol coefficient,are also well known to the skilled artisan. See also the Examples below.

EXAMPLES Exemplary Liquid Compositions

A dry pancreatic enzyme composition containing about 200 USP units/mg ofprotease, about 40 USP units/mg of lipase, and about 250 USP units/mg ofamylase can be diluted with various diluents (water, saline, phosphatebuffered solutions, pH stabilized solutions) and optionally with otheractive or inactive additives (enzyme stabilization systems, buffers,colorants, sanitizers, detergents, disinfectants, antiseptics) to formexemplary liquid compositions for the uses described herein. In someembodiments, the dry enzyme composition can be diluted in a ratio of mgsof the dry enzyme composition to mls of the total diluent in the rangefrom 1 mg enzyme composition:1 ml total diluent to 1 mg enzymecomposition:10,000 mls total diluent, or any value in-between, e.g.,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:50, 1:100, 1:200,1:500, 1:1000, 1:5000, or 1:10,000.

Exemplary Solid Compositions

A dry pancreatic enzyme composition containing about 200 USP units/mg ofprotease, about 40 USP units/mg of lipase, and about 250 USP units/mg ofamylase can be mixed with various active or inactive dry ingredients andadditives (e.g., dry detergents, disinfectants, antiseptics, andsanitizers, such as alkyl ethoxylate sulface, SDS, sodium laurethsulfate, dodecylbenzene, sodium 4-dodecylbenzenesulfonate, enzymestabilization systems, excipients, colorants) to form exemplary solidcompositions. In some embodiments, the dry enzyme composition can bemixed with total mgs of the additives in the range from 1 mg enzymecomposition:1 mg total additives to 1 mg enzyme composition:10,000 mgstotal additives, or any value in-between, e.g., 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:20, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:5000, or1:10,000.

Exemplary Topical Formulations

A dry pancreatic enzyme composition containing about 200 USP units/mg ofprotease, about 40 USP units/mg of lipase, and about 250 USP units/mg ofamylase can be mixed with various carriers appropriate for topicalpharmaceutical formulations in ratios ranging from about 1 mg of theenzyme composition to 1 mg of the carrier to about 1 mg of the enzymecomposition to about 200 mg of the carrier, or any ratio therebetween(e.g., 1:2, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:75, 1:100, 1:150).For example, in one embodiment a 1:25 ratio is employed, and the carrieris petrolatum.

Bacterial Limit Testing—Evaluation of Bacteriocidal and BacteriostaticProperties

The present inventors have surprisingly found in the course of doingbacterial limit testing on a dry pig pancreatic enzyme compositioncomprising about 200 USP units/mg protease activity, about 40 USPunits/mg of lipase activity, and 250 USP units/mg of amylase activitythat E. coli bacteria do not grow in the presence of various dilutionsof this material. Recovery of E. coli CFUs from positive controls“spiked” with E. coli was consistently low to absent. Dilution bacteriallimit testing of the dry composition in both unencapsulated andlipid-encapsulated (20% soy oil by weight of the composition) formdemonstrate minimal to no recovery of the bacterium from positivecontrols to which a known number of CFUs of E. coli had been added. Alack of recovery during such recovery methods suggests a bacteriostaticand/or bacteriocidal nature of the composition.

Methods and Materials

Sample materials—Unencapsulated Porcine Pancreatic Enzyme Concentrate(uPEC) isolated from the pig (Sus scrofa) was manufactured by acommercial supplier (Scientific Protein Labs) to contain approximately200 U/mg protease activity, 40 U lipase activity/mg, and 250 U ofamylase activity/mg. A lipid encapsulated version of this material(ePEC) was obtained by using a modified fluidized bed process and afully hydrogenated highly purified organic oil (fully hydrogenated soyoil) which was then used to coat the enzyme particles at a percentage byweight of about 20% of the final particles.

Methods: Both the ePEC and uPEC underwent standard microbial analysisfor the detection of microbes. Both compositions failed to show anysignificant contamination using Microbial Limits Testing per USPmethods. Additionally, both samples were negative/10 g for Salmonellaspecies and E. coli. Briefly, the procedure for the examination of testcompositions for microbiological suitability (i.e., estimation of thetotal number of viable microorganisms and freedom from specificorganism) is outlined in the USP, chapter 61, “Microbial Limit Tests.”USP 61 outlines preparatory testing, during which is determined the testparameters wherein the test composition itself no longer inhibits themultiplication of viable organisms. USP 1227 (“Validation”) alsoprovides guidance to validate recovery methods. Methods for evaluatingTotal Aerobic Microbial Count, E. Coli, and Salmonella species wereperformed.

Total Aerobic Microbial Count: Preparation of test dilutions—Bulkdilutions of ePEC and uPEC were prepared at 1:10, 1:50, 1;100 and 1:200in Tryptic Soy broth containing 4% Polysorbate 20 and 0.5% lecithin. Thebulk test sample dilution was then split into separate 10 ml aliquots,where were then inoculated with a low number of colony forming units(CFUs<100/mL) of the appropriate microorganisms (S. aureus, P.aeruginosa, E. coli, S. enterica). One millimeter of the inoculatedaliquots was plated in duplicate using the appropriate solid medium(agar). Positive controls were prepared, inoculated, and plated in amanner similar to the test samples. Negative controls were prepared,inoculated with sterile reagent, and plated in a manner similar to thetest samples. The plates were incubated at 30° C. to 35° C. for twodays. At the end of the period the recovery was calculated. Recovery ofinoculated organisms must be at least 70% of the positive control inorder to show no inhibition of growth by the test composition.Triphenyltetrazolium chloride was used to count the plates.

Test for Salmonella species—Dilutions prepared with Lactose brothcontaining 4% polysorbate 20 and 0.5% lecithin were inoculated with<=100 CFU of Salmonella enterica. The inoculated dilutions wereincubated at 30° C. to 35° C. for 24 hours prior to 1 ml beingtransferred to both Selenite Cystine and Tetrahionate broths. Theselective broths were incubated for 18 hours at 30° C. to 35° C. priorto being streaked to brilliant green, bismuth sulfite an xylose lysinedeoxycholate agars. The selective agar plates were incubated at 30° C.to 35° C. for 24 hours. The plates were observed for coloniescharacteristic of Salmonella species. Where observed, a representativecolony was confirmed to be Salmonella species using an API 20ebiochemical identification test.

Test for E. coli—Dilutions prepared with Lactose broth containing 4%polysorbate 20 and 0.5% lecithin were inoculated with <=100 CFU of E.coli. The inoculated dilutions were incubated at 30° C. to 35° C. for 24hours prior to being streaked to MacKonkey agar. The plates wereobserved for colonies characteristic of E. coli. Where observed, arepresentative colony was confirmed to be E. coli using an API 20ebiochemical identification test.

Results

Total Aerobic Count: Test Results using the uncoated PEC (uPEC) areshown in Table 1, below. As shown, for dilutions of 1:50, 1:100, and1:200 the percent recovery of S. aureus was 0%, demonstrating thebacteriocidal and/or bacteriostatic action of uPEC of S. aureus.

TABLE 1 Recovery of Microorganisms following Incubation with uPECNegative Positive Test % Organism Control Control Dilution AverageRecovery S. Aureus 0 CFU 57 CFU 1:50  0 CFU  0% 1:100  0 CFU  0% 1:200 0 CFU  0% E. coli 0 CFU 63 CFU 1:50  0 CFU  0% 1:100  1 CFU  2% 1:200 1 CFU  2% S. enterica 0 CFU 66 CFU 1:50 48 CFU 73% 1:100 62 CFU 94%1:200 56 CFU 85%

The uPEC percent recovery for E. coli is also given in Table 1. For adilution of 1:50 the recovery was 0%, for a dilution of 1:100 therecovery was 2%, and for a dilution of 1:200 the recovery was 2%.

The uPEC percent recovery for S. enterica is also given in Table 1. Fora dilution of 1:50 the recovery was 73%, for a dilution of 1:100 therecovery was 94%, and for a dilution of 1:200 the recovery was 85%.

Test results using the lipid encapsulate PEC (ePEC) are given in Table2, below. As shown, for dilutions of 1:50 the recovery of S. aureus was29%, for a dilution of 1:100 the recovery was 0%, and for a 1:200 thepercent recovery of S. aureus was also 0%.

TABLE 2 Recovery of Microorganisms following Incubation with ePECNegative Positive Test % Organism Control Control Dilution AverageRecovery S. aureus 0 CFU 34 CFU 1:50 10 CFU 29% 1:100  0 CFU  0% 1:200 0 CFU  0% E. coli 0 CFU 38 CFU 1:50 12 CFU 32% 1:100  9 CFU 17% 1:20028 CFU 78% S. enterica 0 CFU 61 CFU 1:50 53 CFU 87% 1:100 N/P N/P 1:200N/P N/P

The ePEC percent recovery for E. Coli are also in Table 2. For adilution of 1:50 the recovery was 32%, for a dilution of 1:100 therecovery was 17%, and for a dilution of 1:200 the recovery was 78%.

The ePEC percent recovery for S. enterica are also given in Table 2. Fora dilution of 1:50 the recovery was 87%, for a dilution of 1:100 and1:200 the tests were not performed.

Positive controls as reported in Tables 1 and 2 clearly demonstrate thatthe growth media for all microbiological cultures were functioningeffectively. In Table 1, it can be seen that the uPEC composition washighly effective on both S. aureus and E. coli. This is substantiated bythe fact that recovery of positively spiked controls failed to meet theUSP criterion of positive recovery (at least 70% of spiked sample CFUsrecovered).

It is also important to note that the bacteriostatic/bactericidalactivity showed species specificity. Samples of S. enterica showedexcellent recovery using both coated and uncoated PEC. Because bacteriashare common cell wall and membrane structures such as lipids andpeptidoglycans, it is possible that these results point to thesensitivity of a more species specific cellular component such as aprotein. If lipase or amylase action alone were sufficient to inducebacterial death or suppression of growth, then it would be unlikely tosee the recovery of S. enterica at such robust levels. It is possiblethat species specific protein(s) in S. aureus and E. coli share similarpeptide sequences and the appropriate local tertiary structure to allowfor enzymatic attack by one or several of the proteases present in PEC,leading to a subsequent destruction of the bacterium. These results donot however, rule out a multi-staged event where action by lipases andamylases against the bacterial cell wall and membrane first exposeextracellular or transmembrane proteins which have the appropriateprimary and tertiary structure to lend themselves to enzymaticdegradation by one or many of the PEC proteases.

These results demonstrate a clear bacteriostatic effect on S. aureus andE. coli for both lipid coated and uncoated PEC. Because this experimentrelies upon an end point read out of bacterial colony growth while thesubject bacteria remain in the presence of PEC, it is not possible onthe basis of these results to rule out the possibility of abacteriostatic effect only. Thus, failure to recover a sufficient numberof viable colony forming units could be the result of the continuedsuppressive presence of the PEC material, as opposed to the induction ofbacterial death. Consequently, additional experimental testing wascarried out in order to more conclusively evaluate the bactericidalcapabilities of PEC.

Materials and Methods: The uncoated PEC formulation described abovepreviously was used to evaluate bacteriocidal activity of theformulations.

Preparation of test dilutions—Bulk dilutions of uPEC were prepared at1:100 and 1:200 in Tryptic Soy broth containing 4% Polysorbate 20 and0.5% lecithin. The bulk dilution was then split into separate 10 mlaliquots, where were then inoculated with a low number of colony formingunits (CFUs<100) of the appropriate microorganisms. One millimeter ofthe inoculated aliquots was plated in duplicate using the appropriatesolid medium (agar). Positive controls were prepared, inoculated, andplated in a manner similar to the test samples. Negative controls wereprepared, inoculated with sterile reagent, and plated in a mannersimilar to the test samples. The plates were incubated at 30° C. to 35°C. for two days. Following this initial incubation, cultured materialwas then collected and washed in Phosphate Buffered Saline (PBS) andfiltered in order to remove PEC. The material was resuspended in dilutedin Tryptic Soy broth as per above and re-plated onto fresh solid (agar)media and incubated for an additional 2 days. At the end of this period,colonies were enumerated and the percentage recovery calculated. At theend of the period the recovery was calculated. Recovery of inoculatedorganisms must be at least 70% in order to show no inhibition of growth.Triphenyltetrazolium chloride was used to count the plates.

Tests for E. coli—Dilutions prepared with Lactose broth containing 4%polysorbate 20 and 0.5% lecithin were inoculated with <=100 CFU of E.coli. The inoculated dilutions were incubated at 30° C. to 35° C. for 24hours prior to being streaked to MacKonkey agar. The plates wereobserved for colonies characteristic of E. coli. Where observed, arepresentative colony was confirmed to be E. coli using an API 20ebiochemical identification test.

The tests were also repeated for both S. Aureus and E. Coli at Dilutionsof 1:20, 1:40, and 1:80.

Test Results

Test Results using the uncoated uPEC are given in Tables 3 and 4, below,demonstrating recovery of bacteria following replating of bacteriaalone, post-incubation with uPEC and washing. As shown in Table 3, fordilutions of 1:100, and 1:200, the percent recovery of S. aureus was 13%and 48%, respectively, clearly demonstrating the bacteriocidal action ofuPEC on S. aureus. As shown in Table 4, the percent recovery of S.Aureus at a dilution of 1:20 is 2%, for a dilution of 1:40 it is 4%, andfor a dilution of 1:80 it is 23%.

TABLE 3 Bactericidal Action Post Wash/Recovery of MicroorganismsNegative Positive Test % Organism Control Control Dilution AverageRecovery S. Aureus 0 CFU 48 CFU 1:100  6 CFU 13% 1:200 23 CFU 48% E.coli 0 CFU 46 CFU 1:100  1 CFU  2% 1:200 13 CFU 28%

TABLE 4 Bactericidal Action Post Wash/Recovery of MicroorganismsNegative Positive Test % Organism Control Control Dilution AverageRecovery S. Aureus 0 CFU 47 CFU 1:20  1 CFU  2% 1:40  2 CFU  4% 1:80 11CFU 23% E. coli 0 CFU 43 CFU 1:20  0 CFU  0% 1:40  0 CFU  0% 1:80  5 CFU12%

The recovery for E. coli is also given in Tables 3 and 4. As shown inTable 3, for a dilution of 1:100 the recovery was 2%, and for a dilutionof 1:200 the recovery was 28%. As shown in Table 4, the percent recoveryof E. Coli at dilution of 1:20 is 0%, for a dilution of 1:40 it is 0%,and for a dilution of 1:80 it is 12%

Positive controls demonstrate that the growth media for allmicrobiological cultures were functioning effectively. This argues for atrue bactericidal effect when the selected organisms are exposed to PEC.If the action of the PEC were merely bacteriostatic, removing the PECfrom co-culture with the test bacteria would result in a release fromany suppressive action by the PEC with a recovery of active growth.However, removal of PEC does not result in any such recovery, eventhough cultures were left to grow over a full 24 hr period. Thisstrongly supports the notion that the reason the selected organisms failto show recovery of CFU's upon exposure to PEC is because of abactericidal action of the PEC. This action could include mechanismssuch as physical and irreversible damage to cell surface lipids,membrane proteins or bacterial capsule by enzymatic degradation, leadingto extracellular and intercellular ionic electrolyte imbalances, changesin acidity, and damage to genetic material.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for the treatment or prevention of E. coli infection in abird or a mammal, comprising administering to the bird or mammal atherapeutically effective amount of a pharmaceutical compositioncomprising one or more digestive enzymes.
 2. The method of claim 1wherein the one or more digestive enzymes comprise one or more enzymesselected from the group consisting of proteases, amylases, celluloses,sucrases, maltases, papain, and lipases.
 3. The method of claim 1wherein the one or more digestive enzymes comprise one or morepancreatic enzymes.
 4. The method of claim 1 wherein the one or more ofthe digestive enzymes comprise pig enzymes.
 5. The method of claim 2wherein the proteases comprise chymotrypsin and trypsin.
 6. The methodof claim 1 wherein the one or more digestive enzymes are, independently,derived from an animal source, a microbial source, a plant source, afungal source, or are synthetically prepared.
 7. The method of claim 1wherein the mammal is a pig, horse, cow, dog, cat, monkey, rat, mouse,sheep, goat or human, or the bird is a chicken, duck, turkey, or goose8. The method of claim 6 where the animal source is a pig pancreas. 9.The method of claim 1 wherein the pharmaceutical composition comprisesat least one amylase, a mixture of proteases comprising chymotrypsin andtrypsin, and at least one lipase.
 10. The method of claim 1 wherein thepharmaceutical composition comprises at least one protease and at leastone lipase, and wherein the ratio of total proteases to total lipases(in USP units) ranges from about 1:1 to about 20:1.
 11. The method ofclaim 11 wherein the ratio of proteases to lipases ranges from about 4:1to about 10:1.
 12. The method of claim 1 wherein the pharmaceuticalcomposition is a dosage formulation selected from the group consistingof: pills, tablets, capsules, caplets, sprinkles, creams, lotions,aerosols, emulsions, powders, liquids, gels, and a combination of anythereof.
 13. The method of claim 1, wherein the pharmaceuticalcomposition is formulated for oral administration.
 14. The method ofclaim 1, wherein the pharmaceutical composition is formulated fortopical administration.
 15. The method of claim 1, wherein thepharmaceutical composition is effective against STEC.
 16. The method ofclaim 1, wherein the pharmaceutical composition is effective againstETEC or EPEC.
 17. A method of treating a mammal or bird exhibiting oneor more symptoms of an E. coli infection comprising administering to themammal or bird a therapeutically effective amount of a compositioncomprising one or more digestive enzymes.
 18. The method of claim 1 or17, further comprising administering a beta-lactam antibiotic to themammal or bird.
 19. A method for treating diarrhea in an individual,comprising administering a pharmaceutical composition comprising one ormore digestive enzymes to the individual.
 20. The method of claim 19,wherein the individual is diagnosed with an E. coli infection
 21. Themethod of claim 20, wherein the E. coli is a STEC.
 22. A method forsanitizing or disinfecting a surface to reduce the amount of E. colithereon or to eradicate the E. coli thereon, comprising applying to thesurface a composition comprising one or more digestive enzymes.
 23. Themethod of claim 22, wherein the surface is a nonliving or inanimatesurface.
 24. The method of claim 23, wherein the surface is on a medicaldevice.
 25. The method of claim 23, wherein the surface is a food.
 26. Amethod for reducing the amount of E. coli present on a skin region,tissue, or wound of a mammal or bird comprising applying to the skinregion, tissue, or wound a composition comprising one or more digestiveenzymes.
 27. A disinfectant comprising one or more digestive enzymes,wherein the disinfectant has a phenol coefficient of >1 to about 20 forS. aureus or E. coli.
 28. An antibiotic comprising one or more digestiveenzymes, wherein the antibiotic is bacteriocidal and/or bacteriostaticfor E. coli.
 29. A detergent comprising one or more digestive enzymes,wherein the detergent is bacteriocidal and/or bacteriostatic for E.coli.
 30. An antiseptic comprising one or more digestive enzymes,wherein the antiseptic is bacteriocidal and/or bacteriostatic for E.coli.
 31. A disinfectant comprising one or more digestive enzymes,wherein the disinfectant is bacteriocidal and/or bacteriostatic for E.coli.